Sugarcane centromere sequences and minichromosomes

ABSTRACT

The invention is generally related to Sugarcane mini-chromosomes and recombinant chromosomes containing Sugarcane centromere sequences. In addition, the invention provides for methods of generating Sugarcane plants transformed with these Sugarcane mini-chromosomes. Sugarcane mini-chromosomes with novel compositions and structures are used to transform Sugarcane cells which are in turn used to generate Sugarcane plants. Methods for generating Sugarcane plants include methods for delivering the Sugarcane mini-chromosomes into Sugarcane cell to transform the cell, methods for selecting the transformed cell, and methods for isolating Sugarcane plants transformed with the Sugarcane mini-chromosome or recombinant chromosome.

RELATED APPLICATION INFORMATION

This application claims the benefit of U.S. Provisional Application No.61/228,019, filed Jul. 23, 2009, the disclosure of which is incorporatedby reference herein in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to Sugarcane mini-chromosomesand recombinant chromosomes containing Sugarcane centromere sequences aswell as Sugarcane cells and plants comprising the same.

BACKGROUND OF THE INVENTION

Two general approaches are used for introduction of new heritablegenetic information (“transformation”) into cells. One approach is tointroduce the new genetic information as part of another DNA molecule,referred to as an “episomal vector,” or “mini-chromosome”, which can bemaintained as an independent unit (an episome) apart from the hostchromosomal DNA molecule(s). Episomal vectors contain all the necessaryDNA sequence elements required for DNA replication and maintenance ofthe vector within the cell. Many episomal vectors are available for usein bacterial cells (for example, see Maniatis et al., “MolecularCloning: a Laboratory Manual,” Cold Spring Harbor Laboratory, ColdSpring Harbor, N. Y. 1982). However, only a few episomal vectors thatfunction in higher eukaryotic cells have been developed. Highereukaryotic episomal vectors were primarily based on naturally occurringviruses. In higher plant systems gemini viruses are double-stranded DNAviruses that replicate through a double-stranded intermediate upon whichan episomal vector could be based, although the gemini virus is limitedto an approximately 800 bp insert. Although an episomal plant vectorbased on the Cauliflower Mosaic Virus has been developed, its capacityto carry new genetic information also is limited (Brisson et al.,Nature, 310:511, 1981).

The other general method of genetic transformation involves integrationof introduced DNA sequences into the recipient cell's chromosomes,permitting the new information to be replicated and partitioned to thecell's progeny as a part of the natural chromosomes. The introduced DNAusually can be broken and joined together in various combinations beforeit is integrated at random sites into the cell's chromosome (see, forexample Wigler et al., Cell, 11:223, 1977). Common problems with thisprocedure are the rearrangement of introduced DNA sequences andunpredictable levels of expression due to the location of the transgeneintegration site in the host genome or so called “position effectvariegation” (Shingo et al., Mol. Cell. Biol., 6:1787, 1986). Further,unlike episomal DNA, integrated DNA cannot normally be preciselyremoved. A more refined form of integrative transformation can beachieved by exploiting naturally occurring viruses that integrate intothe host's chromosomes as part of their life cycle, such as retroviruses(see Chepko et al., Cell, 37:1053, 1984).

One common genetic transformation method used in higher plants is basedon the transfer of bacterial DNA into plant chromosomes that occursduring infection by the phytopathogenic soil bacterium Agrobacterium(see Nester et al., Ann. Rev. Plant Phys., 35:387-413, 1984). Bysubstituting genes of interest for a portion of the naturallytransferred bacterial sequences (called T-DNA), investigators have beenable to introduce new DNA into plant cells. However, even this more“refined” integrative transformation system is limited in three majorways. First, DNA sequences introduced into plant cells using theAgrobacterium T-DNA system are frequently rearranged (see Jones et al.,Mol Gen. Genet., 207:478, 1987). Second, the expression of theintroduced DNA sequences varies between individual transformants (seeJones et al., Embo J., 4:2411-2418, 1985). This variability ispresumably caused by rearranged sequences and the influence ofsurrounding sequences in the plant chromosome (i.e., position effects),as well as methylation of the transgene. Finally, insertion of extraelements into the genome can disrupt the genes, promoters or othergenetic elements necessary for normal plant growth and function.

Another widely used technique to genetically transform plants involvesthe use of microprojectile bombardment to integrate DNA sequences intothe genome. In this process, a nucleic acid containing the desiredgenetic elements to be introduced into the plant's native chromosome isdeposited on or in small metallic particles, e.g., tungsten, platinum,or preferably gold, which are then delivered at a high velocity into theplant tissue or plant cells. However, similar problems arise as withAgrobacterium-mediated gene transfer, and as noted above expression ofthe inserted DNA can be unpredictable and insertion of extra elementsinto the genome can disrupt and adversely impact plant processes.

One attractive alternative to the commonly used methods oftransformation is the use of an artificial chromosome. Artificialchromosomes are episomal nucleic acid molecules that exist autonomouslyfrom the native chromosomes of the host genome. They can be linear orcircular DNA molecules that are comprised of cis-acting nucleic acidsequence elements that provide replication and partitioning activities(see Murray et al., Nature, 305:189-193, 1983). Desired elementsinclude: (1) origin of replication, which are the sites for initiationof DNA replication, (2) centromeres (site of kinetochore assembly andresponsible for proper distribution of replicated chromosomes intodaughter cells at mitosis or meiosis), and (3) if the chromosome islinear, telomeres (specialized DNA structures at the ends of linearchromosomes that function to stabilize the ends and facilitate thecomplete replication of the extreme termini of the DNA molecule). Anadditional desired element is a chromatin organizing sequence. It iswell documented that centromere function is crucial for stablechromosomal inheritance in almost all eukaryotic organisms (reviewed inNicklas 1988). The centromere accomplishes this by attaching, viacentromere binding proteins, to the spindle fibers during mitosis andmeiosis, thus ensuring proper gene segregation during cell divisions.

Artificial chromosomes have been engineered using one of two approaches.The first approach identifies and assembles the desired chromosomalelements into an artificial construct. This approach has been describedas “bottom-up” and involves the use of a heterologous system (i.e.bacteria or fungal) to perform the various cloning steps necessary toassemble the artificial chromosome. Artificial chromosomes of this typewill be referred to in this application as “mini-chromosomes”. Thesecond approach derives the artificial chromosome from existingchromosomes through chromosome fragmentation and, optionally, subsequentaddition of desired elements including transgenes. For example, anexisting chromosome can be induced to undergo breakage events thatresult in chromosomal fragments. Minimal fragments that possess theelements for replication and segregation during cell division (e.g.centromere, origins of replication and/or telomeres) can be identified.These derived artificial chromosomes can then be used as targets forfurther manipulation including the addition of one or more transgenes.This approach has been described as “top-down” and does not require theuse of a heterologous system (e.g. bacterial or fungal) since it doesn'trequire in vitro-based cloning steps. Artificial chromosomes of thistype will be referred to in this application as “recombinantchromosomes”.

The essential chromosomal elements for construction of artificialchromosomes have been precisely characterized in lower eukaryoticspecies, and more recently in mouse and human. Autonomous replicationsequences (ARSs) have been isolated from unicellular fungi, includingSaccharomyces cerevisiae (brewer's yeast) and Schizosaccharomyces pombe(see Stinchcomb et al., 1979 and Hsiao et al., 1979). An ARS behaveslike an origin of replication allowing DNA molecules that contain theARS to be replicated in concert with the rest of the genome afterintroduction into the cell nuclei of these fungi. DNA moleculescontaining these sequences replicate, but in the absence of a centromerethey are not partitioned into daughter cells in a controlled fashionthat ensures efficient chromosome inheritance.

Artificial chromosomes have been constructed in yeast using the threecloned essential chromosomal elements (see Murray et al., Nature,305:189-193, 1983). None of the essential components identified inunicellular organisms, however, function in higher eukaryotic systems.For example, a yeast centromere sequence will not confer stableinheritance upon vectors transformed into higher eukaryotes.

In contrast to the detailed studies done in yeast, less is known aboutthe molecular structure of functional centromeric DNA of highereukaryotes. Ultrastructural studies indicate that higher eukaryotickinetochores, which are specialized complexes of proteins that form onthe centromere during late prophase, are large structures (mammaliankinetochore plates are approximately 0.3 μm in diameter) which possessmultiple microtubule attachment sites (reviewed in Rieder, 1982). It istherefore possible that the centromeric DNA regions of these organismswill be correspondingly large, although the minimal amount of DNAnecessary for centromere function may be much smaller.

While the above studies have been useful in elucidating the structureand function of centromeres, it was not known whether informationderived from lower eukaryotic or mammalian higher eukaryotic organismswould be applicable to Sugarcane. There exists a need for clonedcentromeres from Sugarcane, which would represent a first step in theproduction of Sugarcane artificial chromosomes, or in the identificationof Sugarcane recombinant chromosomes. There further exists a need forSugarcane cells, plants, seeds and progeny containing functional,stable, and autonomous artificial or recombinant chromosomes capable ofcarrying a large number of different genes and genetic elements.

SUMMARY OF THE INVENTION

In one aspect, the present invention addresses Sugarcanemini-chromosomes comprising a Sugarcane centromere having one or morerepeated nucleotide sequences, described in further detail herein. Insome embodiments, such mini-chromosomes comprise a centromere comprisingone or more selected repeated nucleotide sequences derived fromSugarcane, including those isolated from Sugarcane genomic DNA andsynthetic arrays of repeat sequences. In other embodiments, theinvention addresses Sugarcane recombinant chromosomes.

In another aspect, the invention provides modified or “adchromosomal”Sugarcane plants, containing functional, stable, autonomousmini-chromosomes or recombinant chromosomes.

The invention provides for isolated Sugarcane mini-chromosomescomprising a centromere, wherein the centromere comprises at least twocopies of a repeated nucleotide sequence(s), and wherein the centromereconfers the ability to segregate to daughter cells. The repeatednucleotide sequence(s) may be short Sugarcane satellite sequences suchas those sequences set out as SEQ ID NOS: 1-201, the consensus Sugarcanesatellite sequence set out as SEQ ID NO: 202, or the block of Sugarcanesatellite sequence set out as SEQ ID NO:204. The repeated nucleotidesequences may be longer sequences such as the Sugarcane retrotransposonsequence CRS, set out as SEQ ID NO: 203.

The invention also provides cells comprising a polynucleotide, nucleicacid, vector, Sugarcane centromere, Sugarcane artificial mini-chromosomeand/or Sugarcane recombinant chromosome of the invention. Inembodiments, the cell is an isolated cell. In other representativeembodiments, the cell is a Sugarcane cell.

Accordingly, as one aspect, the invention provides a polynucleotidecomprising a nucleotide sequence selected from the group consisting of(a) the nucleotide sequence of any of SEQ ID NOS: 1-204; (b) anucleotide sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%or 99%/identical to the nucleotide sequence of any of SEQ ID NOS: 1-204,optionally wherein the nucleotide sequence is functional as a sugar caneplant centromere (e.g., confers the ability to segregate to a daughtercell); and/or (c) a nucleotide sequence that hybridizes to thenucleotide sequence of any of SEQ ID NOS: 1-204 under stringentconditions comprising hybridization at 65° C. and washing three timesfor 15 minutes with 0.25×SSC, 0.1% SDS at 65° C., optionally wherein thenucleotide sequence is functional as a sugar cane plant centromere.

As another aspect, the invention provides a nucleic acid comprising anarray comprising at least about two, at least about ten, at least about100, from about 2 to about 1000, or from about 5 to about 250 copies ofa polynucleotide of the invention. In representative embodiments, thearray is from about 1 to about 200 kb in length, optionally from about15 to about 28 kb in length. In exemplary embodiments, the nucleic acidis functional as a sugar cane plant centromere.

The invention further encompasses a Sugarcane centromere comprising apolynucleotide or nucleic acid of the invention.

Also provided by the invention is a Sugarcane artificial chromosomecomprising a polynucleotide, nucleic acid or centromere of theinvention. In embodiments of the invention, the Sugarcane artificialchromosome further comprises an exogenous nucleic acid (e.g., at leastthree exogenous nucleic acids), at least one of which may optionally belinked to a heterologous regulatory sequence functional in Sugarcaneplant cells.

In further representative embodiments, the Sugarcane artificialchromosomes of the invention exhibit a mitotic segregation efficiency inSugarcane plant cells of at least about 60%, 70%, 80%, 85%, 90%, 95% ormore.

Also encompassed by the present invention is a vector comprising apolynucleotide, nucleic acid, Sugarcane centromere, or Sugarcaneartificial chromosome of the invention.

As still another aspect, the invention provides a cell comprising apolynucleotide, nucleic acid, Sugarcane centromere, Sugarcane artificialchromosome, or vector of the invention. In representative embodiments,the cell is a Sugarcane plant cell.

In particular embodiments, the invention provides a Sugarcane plant cellcomprising a Sugarcane artificial chromosome, wherein the Sugarcaneartificial chromosome is not integrated into the genome of the Sugarcaneplant cell. The Sugarcane plant cell can optionally comprise a Sugarcaneartificial chromosome that comprises an exogenous nucleic acid, whereinthe Sugarcane plant cell exhibits an altered phenotype associated withthe expression of the exogenous nucleic acid. The altered phenotype canbe any phenotypic change of interest that can be detected and,optionally, measured. In an exemplary embodiment, the altered phenotypecomprises altered expression (e.g., increased or decreased expression)of a native gene. In other embodiments, the altered phenotype comprisesaltered expression of an exogenous gene.

As yet a further aspect, the invention provides a Sugarcane planttissue, a Sugarcane plant, and/or a Sugarcane plant part comprising aSugarcane plant cell of the invention.

The invention also provides a Sugarcane seed obtained from a Sugarcaneplant of the invention.

The invention also contemplates a Sugarcane plant progeny comprising aSugarcane artificial chromosome, wherein the plant progeny is the resultof breeding a Sugarcane plant of the invention that comprises thesugarcane artificial chromosome.

As still another aspect, the invention provides a method of using aSugarcane plant of the invention, wherein the Sugarcane plant comprisesa Sugarcane artificial chromosome comprising an exogenous nucleic acidencoding a recombinant protein, the method comprising growing the plantto produce the recombinant protein. The method can optionally furthercomprise the step of harvesting and/or processing the Sugarcane plant.

In exemplary embodiments, the invention provides for a Sugarcane plantcell comprising a Sugarcane mini-chromosome comprising a Sugarcanecentromere that comprises at least two repeat nucleotide sequences thathave a sequence that hybridizes under conditions comprisinghybridization at 65° C. and washing three times for 15 minutes with0.25×SSC, 0.1% SDS at 65° C. to a nucleotide sequence selected from thegroup consisting of SEQ ID NOS: 1-204, and wherein the centromereconfers the ability to segregate to daughter Sugarcane cells.Alternatively, the hybridization conditions may comprise hybridizationat 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDSat 65° C.

In another exemplary embodiment, the invention provides for a Sugarcaneplant cell comprising a Sugarcane mini-chromosome comprising a Sugarcanecentromere, wherein the centromere comprises at least two copies of arepeated nucleotide sequence(s) that has a sequence that is at least 80%identical to a nucleotide sequence selected from the group consisting ofSEQ ID NOS: 1-204, and wherein the centromere confers the ability tosegregate to daughter Sugarcane cells. The invention also provides for aSugarcane plant cell comprising a Sugarcane mini-chromosome wherein therepeated nucleotide sequence comprises a sequence that is at least 85%identical, or 90% identical, or 95% identical or 98% identical to anucleotide sequence selected from the group consisting of SEQ ID NOS:1-204.

In another embodiment, the invention provides for a Sugarcane plant cellcomprising a Sugarcane Applied Mini-chromosome comprising at least twocopies of a repeated nucleotide sequence that is at least 80% identicalto the nucleotide sequence of any one of SEQ ID NOS: 1-204 or hybridizesto the nucleotide sequence of any one of SEQ ID NOS: 1-204 understringent conditions comprising hybridization at 65° C. and washingthree times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C., and aTransgene Expression Cassette.

In a further embodiment, the invention provides for a Sugarcane plantcell comprising a Sugarcane mini-chromosome comprising a Sugarcanecentromere, wherein the centromere comprises (a) at least two copies ofa Sugarcane satellite nucleotide sequence (e.g., SEQ ID NO:204), and (b)at least two copies of a Sugarcane CRS nucleotide sequence (SEQ ID NO:203), and wherein the centromere confers the ability to segregate todaughter Sugarcane cells. In another embodiment, the invention providesfor a Sugarcane plant cell comprising a Sugarcane mini-chromosomecomprising a Sugarcane centromere, wherein the centromere comprises (a)at least one array of Sugarcane satellite nucleotide sequences, and (b)at least one array of a Sugarcane CRS nucleotide sequence (SEQ ID NO:203), and wherein the centromere confers the ability to segregate todaughter Sugarcane cells. The Sugarcane satellite nucleotide sequencemay be one of the sequences set out as SEQ ID NOS: 1-202 or SEQ ID NO:204, or a sequence that hybridizes under conditions comprisinghybridization at 65° C. and washing three times for 15 minutes with0.25×SSC, 0.1% SDS at 65° C. to a nucleotide sequence selected from thegroup consisting of SEQ ID NOS: 1-202 and SEQ ID NO: 204, or a sequencethat is at least 80% identical to a nucleotide sequence selected fromthe group consisting of SEQ ID NOS: 1-202 and SEQ ID NO: 204.

In addition, the invention provides for a Sugarcane plant cellcomprising a Sugarcane Applied Mini-chromosome comprising a Sugarcanecentromere, wherein the Sugarcane centromere comprises (a) at least 5copies of a repeated nucleotide sequence within 1 kb of nucleotidesequence, wherein the repeated nucleotide sequence is at least 80%identical to the nucleotide sequence of any one of SEQ ID NOS: 1-202 orSEQ ID NO: 204 or hybridizes to the nucleotide sequence of any one ofSEQ ID NOS: 1-202 or SEQ ID NO: 204 under stringent conditionscomprising hybridization at 65° C. and washing three times for 15minutes with 0.25×SSC, 0.1% SDS at 65° C., and (b) at least 2 copies ofa repeated nucleotide sequence that is at least 80% identical over itslength to the nucleotide sequence of any one of SEQ ID NO: 203 orhybridizes to the nucleotide sequence of any one of SEQ ID NO: 203 understringent conditions comprising hybridization at 65° C. and washingthree times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C.

In another embodiment, the invention provides a Sugarcane plant cellcomprising (a) a polynucleotide sequence that is transcribed as a firstRNA, (b) a polynucleotide sequence that is transcribed as a second RNA,and (c) a polynucleotide sequence that is transcribed as a third RNA,wherein transcription of the polynucleotide sequences results inincreased biomass of a Sugarcane plant.

In an additional embodiment, the invention provides for a Sugarcaneplant cell comprising a transgene expression cassette not integratedinto the plant cell genome, wherein the Transgene Expression Cassettecomprises (a) a polynucleotide sequence that is transcribed as a firstRNA, (b) a polynucleotide sequence that is transcribed as a second RNA,and (c) a polynucleotide sequence that is transcribed as a third RNA,wherein transcription of the polynucleotide sequences results inincreased biomass of a Sugarcane plant.

The invention provides for a Sugarcane plant cell comprising arecombinant chromosome comprising at least two copies of a repeatednucleotide sequence(s), and wherein the centromere confers the abilityto segregate to daughter cells. The repeated nucleotide sequence(s) maybe short Sugarcane satellite sequences such as those sequences set outas SEQ ID NOS: 1-201, the consensus Sugarcane satellite sequence set outas SEQ ID NO: 202, or the block of Sugarcane satellite sequence set outas SEQ ID NO: 204. The repeated nucleotide sequences may be longersequences such as the Sugarcane retrotransposon sequence CRS, set out asSEQ ID NO: 203.

In exemplary embodiments, the invention provides for a Sugarcane plantcell comprising a recombinant chromosome comprising a Sugarcanecentromere that comprises at least two repeat nucleotide sequences thathave a sequence that hybridizes under conditions comprisinghybridization at 65° C. and washing three times for 15 minutes with0.25×SSC, 0.1% SDS at 65° C. to a nucleotide sequence selected from thegroup consisting of SEQ ID NOS: 1-204, and wherein the centromereconfers the ability to segregate to daughter Sugarcane cells.Alternatively, the hybridization conditions may comprise hybridizationat 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDSat 65° C.

In another exemplary embodiment, the invention provides for a Sugarcaneplant cell comprising a recombinant chromosome comprising at least twocopies of a repeated nucleotide sequence(s) that has a sequence that isat least 80% identical to a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 1-204, and a transgene expression cassettecomprising at least three exogenous nucleic acids. The invention alsoprovides for Sugarcane recombinant chromosomes wherein the repeatednucleotide sequence comprise a sequence that is at least 85% identical,or 90% identical, or 95% identical or 98% identical to a nucleotidesequence selected from the group consisting of SEQ ID NOS: 1-204.

In a further embodiment, the invention provides for a Sugarcane plantcell comprising a Sugarcane recombinant chromosome comprising aSugarcane centromere, wherein the centromere comprises (a) at least twocopies of a Sugarcane satellite nucleotide sequence, and (b) at leasttwo copies of a Sugarcane CRS nucleotide sequence (SEQ ID NO: 203), andwherein the centromere confers the ability to segregate to daughterSugarcane cells. In another embodiment, the invention provides for aSugarcane recombinant chromosome comprising a Sugarcane centromere,wherein the centromere comprises (a) at least one array of Sugarcanesatellite nucleotide sequences, and (b) at least one array of aSugarcane CRS nucleotide sequence (SEQ ID NO: 203), and wherein thecentromere confers the ability to segregate to daughter Sugarcane cells.The Sugarcane satellite nucleotide sequence may be one of the sequencesset out as SEQ ID NOS: 1-202 or SEQ ID NO: 204, or a sequence thathybridizes under conditions comprising hybridization at 65° C. andwashing three times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C. toa nucleotide sequence selected from the group consisting of SEQ ID NOS:1-202 and SEQ ID NO: 204, or a sequence that is at least 80% identicalto a nucleotide sequence selected from the group consisting of SEQ IDNOS: 1-202 and SEQ ID NO: 204.

Alternatively, the invention provides for Sugarcane plant cellscomprising a recombinant chromosome that has not been maintained in acell of a heterologous organism.

In another embodiment, the invention provides for a Sugarcane plant cellcomprising (a) at least two copies of a repeated nucleotide sequencethat is at least 80% identical to the nucleotide sequence of any one ofSEQ ID NOS: 1-204 or hybridizes to the nucleotide sequence of any one ofSEQ ID NOS: 1-204 under stringent conditions comprising hybridization at65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDS at65° C., and (b) a Transgene Expression Cassette comprising at leastthree exogenous nucleic acids, wherein the nucleotide sequence and theTransgene Expression Cassette are not integrated into the genome of theSugarcane plant cell.

The invention also provides for a Sugarcane plant cell comprising aSugarcane mini-chromosome comprising a Sugarcane centromere, wherein thecentromere comprises at least two synthetic repeat sequences or asynthetic array of repeated nucleotide sequence, wherein the arraycomprises at least two copies of a repeated nucleotide sequence, andwherein the centromere confers the ability to segregate to daughterSugarcane cells. These artificially synthesized repeated nucleotidesequences may be based on sequence information from natural Sugarcanecentromere sequences, combinations or fragments of natural Sugarcanecentromere sequences including a combination of repeats of differentlengths, a combination of different sequences, a combination of bothdifferent repeat lengths and different sequences, a combination ofdifferent artificially synthesized sequences or a combination of naturalSugarcane centromere sequence(s) and artificially synthesized Sugarcanesequence(s). The polynucleotides comprising synthetic arrays ofSugarcane repeat sequences and synthetic arrays of Sugarcane repeatsequences may be generated using any technique known in the artincluding PCR from Sugarcane genomic DNA (or a clone thereof) or bycustom oligonucleotide synthesis.

The invention provides for any of the Sugarcane mini-chromosomes orrecombinant chromosomes described herein having a centromere comprisingan array of repeated nucleotide sequence that ranges from about 1 kb toabout 200 kb in length, 1 kb to about 100 kb in length, about 1 kb toabout 10 kb in length, about 2 kb to about 12 kb in length, about 5 kbto about 25 kb in length, about 10 kb to about 50 kb in length, about 25kb to 100 kb in length.

The invention further contemplates any of the Sugarcane mini-chromosomesor recombinant chromosomes of the invention having centromerescomprising at least 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 750 bp, 1kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 10.5 kb, 11kb, 11.5 kb, 12 kb, 12.5 kb, 13 kb, 13.5 kb, 14 kb, 14.5 kb, 15 kb, 16kb, 17 kb, 18 kb, 19 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 110 kb, 120 kb, 130 kb, 140 kb,150 kb, 160 kb, 170 kb, 180 kb, 190 kb, 200 kb, 225 kb, 250 kb, 275 kb,300 kb, 325 kb, 350 kb or 375 kb.

In another embodiment, any of the Sugarcane mini-chromosomes orrecombinant chromosomes of the invention comprise centromeres having ncopies of a repeated nucleotide sequence, wherein n is less than 2000,less than 1500, less than 1000, less than 500, less than 400, less than300, less than 250, less than 200, less than 100, less than 90, lessthan 80, less than 70, less than 60, less than 50, less than 40, lessthan 30, less than 25, less than 20, less than 15, less than 10, lessthan 9, less than 8, less than 7, less than 6 or less than 5. Inexemplary embodiments, the centromeres of the Sugarcane mini-chromosomesof the invention comprise n copies of a repeated nucleotide sequence,wherein n is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,550, 600, 650, 700, 750, 800, 850, 900 or 1000. In additional exemplaryembodiments, the centromeres of the Sugarcane mini-chromosomes orrecombinant chromosomes of the invention comprise n copies of a repeatednucleotide sequence where n ranges from 2 to 10, 2 to 20, 2 to 50, 2 to100, 2 to 250, 2 to 500, 2 to 1000, 5 to 15, 5 to 25, 5 to 50, 5 to 100,5 to 250, 5 to 500, 5 to 1000, 15 to 25, 15 to 50, 15 to 100, 15 to 250,15 to 500, 15 to 1000, 25 to 50, 25 to 100, 25 to 250, 25 to 500, 25 to1000, 50 to 100, 50 to 250, 50 to 500, 50 to 1000, 100 to 250, 100 to500, 100 to 1000, 250 to 500, 250 to 1000, or 500 to 1000.

In an embodiment of the invention, any of the Sugarcane mini-chromosomesor recombinant chromosomes of the invention comprise a centromere havingat least 5 consecutive repeated nucleotide sequences (e.g., SEQ ID NO:204) in “head to tail orientation.” In an embodiment of the invention,any of the Sugarcane mini-chromosomes or recombinant chromosomes of theinvention comprise a centromere having at least 5 consecutive repeatednucleotide sequences in “tandem,” in which one repeat sequence isimmediately adjacent to another repeat sequence in any orientation, e.g.head to tail, tail to tail, or head to head. The invention also providesfor any of the Sugarcane mini-chromosomes or recombinant chromosomes ofthe invention comprising a centromere having at least 5 repeatednucleotide sequences that are consecutive. The term “consecutive” refersto the same or similar repeated nucleotide sequences (e.g., at least 70%identical) that follow one after another without being interrupted byother significant sequence elements. Consecutive repeated nucleotidesequences may be in any orientation, e.g. head to tail, tail to tail, orhead to head, and need not be directly adjacent to each other (e.g., maybe 1-50 bp apart).

The invention further provides for any of the Sugarcane mini-chromosomesor recombinant chromosomes of the invention comprising a centromerehaving at least 5 of the consecutive repeated nucleotide sequences(e.g., SEQ ID NO: 204) separated by less than n number of nucleotides,wherein n ranges from 1 to 10, or 1 to 20, or 1 to 30, or 1 to 40, or 1to 50 or wherein n is less than 10 bp or n is less than 20 bp or n isless than 30 bp or n is less that 40 bp or n is less than 50 bp.

The invention also provide for any of the Sugarcane mini-chromosomes orrecombinant chromosomes of the invention comprising a centromere havingat least two arrays of consecutive repeated nucleotide sequences (e.g.,SEQ ID NO: 204), wherein the array comprises at least 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250,300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 or 2000 repeatednucleotide sequences. The repeats within an array may be in tandem inany orientation, e.g. head to tail, tail to tail, or head to head, orconsecutive in any orientation, e.g. head to tail, tail to tail, or headto head. The arrays may be separated by less than n number ofnucleotides, wherein n ranges from 1 to 10, or 1 to 20, or 1 to 30, or 1to 40, or 1 to 50, or 1 to 60, or 1 to 70, or 1 to 80, or 1 to 90, or 1to 100, or wherein n is less than 10 bp or n is less than 20 hp or n isless than 30 bp or n is less that 40 bp or n is less than 50 bp. The twoarrays may comprise the same repeated nucleotide sequence or twodifferent repeated nucleotide sequences (i.e. the first array can becomprised of repeat type 1 and the second array can be comprised ofrepeat type 2—here “type 1” and “type 2” are arbitrary designations).

In one embodiment, the Sugarcane mini-chromosomes or recombinantchromosomes of the invention are 1000 kb or less in length, 900 kb orless in length, 800 kb or less in length or 700 kb or less in length. Inexemplary embodiments, the Sugarcane mini-chromosome is 600 kb or lessin length, 500 kb or less in length, 250 kb or less in length, 100 kb orless in length, 50 kb or less in length, 10 kb or less in length, 5 kbor less in length, or 1 kb or less in length. For example, the Sugarcanemini-chromosomes of the invention are 50 to 250 kb in length, 50 to 100kb in length, 50 to 75 kb in length, 50 to 100 kb in length, 60 kb to 85kb in length, 70 to 90 kb in length, 75 to 100 kb in length, 100 to 250kb in length, 250 to 500 kb in length, 500 to 1000 kb in length. In anexemplary embodiment, the Sugarcane mini-chromosome is 28 kb in length,42 kb in length, 82 kb in length, 87 kb in length, 88 kb in length, 97kb in length, 130 kb in length, 150 kb in length, 200 kb in length orranges from 28-200 kb in length. The mini-chromosome of the inventionpreferably has a segregation efficiency during mitotic division of atleast 60%, at least 80%, at least 90% or at least 95% and/or atransmission efficiency during meiotic division of, e.g., at least 60%,at least 80%, at least 85%, at least 90% or at least 95%.

The Sugarcane mini-chromosome or recombinant chromosomes of theinvention preferably has a segregation efficiency during mitoticdivision of at least 60%, at least 80%, at least 90% or at least 95%and/or a transmission efficiency during meiotic division of, e.g., atleast 60%, at least 80/%, at least 85%, at least 90% or at least 95%.

In another embodiment, the Sugarcane mini-chromosomes or recombinantchromosomes of the invention comprise a site for site-specificrecombination.

The invention also provides for a Sugarcane mini-chromosome, wherein themini-chromosome is derived from a donor clone or a centromere clone andhas substitutions, deletions, insertions, duplications or arrangementsof one or more nucleotides in the mini-chromosome compared to thenucleotide sequence of the donor clone or centromere clone. In oneembodiment, the Sugarcane mini-chromosome is obtained by passage of theSugarcane mini-chromosome through one or more hosts. In anotherembodiment, the mini-chromosome is obtained by passage of themini-chromosome through two or more different hosts. The host may beselected from the group consisting of viruses, bacteria, yeasts. Inanother embodiment, the Sugarcane mini-chromosome is obtained from adonor clone by in vitro methods that introduce sequence variation duringtemplate-based replication of the donor clone, or its complementarysequence. In one embodiment this variation may be introduced by aDNA-dependent DNA polymerase. In a further embodiment a Sugarcaneminichromosome derived by an in vitro method may be further modified bypassage of the mini-chromosome through one or more hosts.

The invention also provides for a Sugarcane mini-chromosome orrecombinant chromosome, wherein the mini-chromosome comprises at leastone exogenous nucleic acid. In further exemplary embodiments, theSugarcane mini-chromosome or recombinant chromosome comprises at leasttwo or more, at least three or more, at least four or more, at leastfive or more, at least ten or more, at least 20 or more, at least 30 ormore, at least 40 or more, at least 50 or more exogenous nucleic acids.

In one embodiment, at least one exogenous nucleic acid of any of theSugarcane mini-chromosomes or recombinant chromosomes of the inventionis operably linked to a heterologous regulatory sequence functional inplant cells, including but not limited to a plant regulatory sequence.The invention also provides for exogenous nucleic acids linked to anon-plant regulatory sequence, such as an arthropod, viral, bacterial,vertebrate or yeast regulatory sequence. The invention also provides forexogenous nucleic acids linked to a regulatory sequence from Sugarcane.

The invention also provides for a mini-chromosome or recombinantchromosome comprising a gene or group of genes that act to improve thetotal recoverable sugar from Sugarcane. Such genes may act to increasethe sugar concentration of the stem juice, increase the amount of juice,increase the stem strength to improve yield, or increase total biomassof the plant. Such genes may be derived from bacterial sequences such asa sucrose isomerase or from animal, plant fungal, or protist sequences.Such genes from plants may include genes involved in sugar metabolism ortransport or genes of unknown function or genes not known to beassociated with sugar metabolism or transport but that have been shownto quantitatively increase total recoverable sugar. Such genes may alsoinclude genes that affect plant height, stem diameter, water metabolismor total biomass. Such genes may also include those that regulate theequilibrium between starch and sugar. Several genes have been shown toimprove sugar accumulation. For example, expression of a bacterialsucrose isomerase can increase Sugarcane sugar content by as much astwo-fold (Birch, R. G., and Wu, L. (2007).

Doubled sugar content in Sugarcane plants modified to produce a sucroseisomer. Plant Biotechnology Journal 5: 109-117). The lignin-deficient“brown midrib” mutations improve sorghum sugar content via their effectson lignin; this phenotype is caused by mutations in cinnamyl alcoholdehydrogenase (CAD), and 14 CAD-like genes are present in the sorghumgenome (Saballos, A., Ejeta, G., Sanche, E., Kang, C., and Vermerris, W.(2008). A Genome-Wide Analysis of the Cinnamyl Alcohol DehydrogenaseFamily in Sorghum (Sorghum bicolor (L.) Moench) Identifies SbCAD2 as theBrown midrib6 Gene. Genetics).

In another embodiment, the Sugarcane mini-chromosome or recombinantchromosome comprises an exogenous nucleic acid that comprises a QTL thatconfers a desirable trait. QTLs that affect total recoverable sugarshave been mapped in Sugarcane (Murray, S. C., Sharma, A., Rooney, W. L.,Klein, P., Mullet, J. E., Mitchell, S. E., Kresovitch, S. (2008) GeneticImprovement of Sorghum as a Biofuel Feedstock: I. QTL for Stem Sugar andGrain Nonstructural Carbohydrates. Crop Sci. 48:2165-2179).

In another embodiment, the Sugarcane mini-chromosome or recombinantchromosome comprises an exogenous nucleic acid that confers herbicideresistance, insect resistance, disease resistance, or stress resistanceon the Sugarcane plant. The invention provides for Sugarcanemini-chromosomes or recombinant chromosomes comprising an exogenousnucleic acid that confers resistance to phosphinothricin or glyphosateherbicide. Nonlimiting examples include an exogenous nucleic acid thatencodes a phosphinothricin acetyltransferase, glyphosateacetyltransferase, acetohydroxyadic synthase or a mutantenoylpyruvylshikimate phosphate (EPSP) synthase. Nonlimiting examples ofexogenous nucleic acids that confer insect resistance include a Bacillusthuringiensis toxin gene or Bacillus cereus toxin gene. In relatedembodiments, the Sugarcane mini-chromosome or recombinant chromosomecomprises an exogenous nucleic acid conferring herbicide resistance, anexogenous nucleic acid conferring insect resistance, and optionally atleast one additional exogenous nucleic acid.

The invention further provides for Sugarcane mini-chromosomes orrecombinant chromosomes comprising additional copies of genes alreadyfound in the Sugarcane genome. The invention also provides for theadditional copies of Sugarcane genes carried on the Sugarcanemini-chromosome or recombinant chromosomes to be operably linked toeither their native regulatory sequences or to heterologous regulatorysequences.

The invention further provides for Sugarcane mini-chromosomes orrecombinant chromosomes comprising an exogenous nucleic acid thatconfers resistance to drought, heat, chilling, freezing, excessivemoisture, ultraviolet light, ionizing radiation, toxins, pollution,mechanical stress or salt stress. The invention also provides for aSugarcane mini-chromosome that comprises an exogenous nucleic acid thatconfers resistance to a virus, bacteria, fungi or nematode.

The invention provides for Sugarcane mini-chromosomes or recombinantchromosomes comprising an exogenous nucleic acid selected from the groupconsisting of a nitrogen fixation gene, a plant stress-induced gene, anutrient utilization gene, a gene that affects plant pigmentation, agone that encodes an antisense or ribozyme molecule, a gene encoding asecretable antigen, a toxin gene, a receptor gene, a ligand gene, a seedstorage gene, a hormone gene, an enzyme gene, an interleukin gene, aclotting factor gene, a cytokine gene, an antibody gene, a growth factorgene, a transcription factor gene, a transcriptional repressor gene, aDNA-binding protein gene, a recombination gene, a DNA replication gene,a programmed cell death gene, a kinase gene, a phosphatase gene, a Gprotein gene, a cyclin gene, a cell cycle control gene, a gene involvedin transcription, a gene involved in translation, a gene involved in RNAprocessing, a gene involved in RNAi, an organellar gene, a intracellulartrafficking gene, an integral membrane protein gene, a transporter gene,a membrane channel protein gene, a cell wall gene, a gene involved inprotein processing, a gene involved in protein modification, a geneinvolved in protein degradation, a gene involved in metabolism, a geneinvolved in biosynthesis, a gene involved in assimilation of nitrogen orother elements or nutrients, a gene involved in controlling carbon flux,a gene involved in respiration, a gene involved in photosynthesis, agene involved in light sensing, a gene involved in organogenesis, a geneinvolved in embryogenesis, a gene involved in differentiation, a geneinvolved in meiotic drive, a gene involved in self incompatibility, agene involved in development, a gene involved in nutrient, metabolite ormineral transport, a gene involved in nutrient, metabolite or mineralstorage, a calcium-binding protein gene, or a lipid-binding proteingene.

The invention also provides for a Sugarcane mini-chromosome orrecombinant chromosome comprising an exogenous enzyme gene selected fromthe group consisting of a gene that encodes an enzyme involved inmetabolizing biochemical wastes for use in bioremediation, a gene thatencodes an enzyme for modifying pathways that produce secondary plantmetabolites, a gene that encodes an enzyme that produces apharmaceutical, a gene that encodes an enzyme that improves changes inthe nutritional content of a plant, a gene that encodes an enzymeinvolved in vitamin synthesis, a gene that encodes an enzyme involved incarbohydrate, polysaccharide or starch synthesis, a gene that encodes anenzyme involved in mineral accumulation or availability, a gene thatencodes a phytase, a gene that encodes an enzyme involved in fatty acid,fat or oil synthesis, a gene that encodes an enzyme involved insynthesis of chemicals or plastics, a gene that encodes an enzymeinvolved in synthesis of a fuel, a gene that encodes an enzyme involvedin synthesis of a fragrance, a gene that encodes an enzyme involved insynthesis of a flavor, a gene that encodes an enzyme involved insynthesis of a pigment or dye, a gene that encodes an enzyme involved insynthesis of a hydrocarbon, a gene that encodes an enzyme involved insynthesis of a structural or fibrous compound, a gene that encodes anenzyme involved in synthesis of a food additive, a gene that encodes anenzyme involved in synthesis of a chemical insecticide, a gene thatencodes an enzyme involved in synthesis of an insect repellent, or agene controlling carbon flux in a plant.

In another embodiment of the invention, any of the Sugarcanemini-chromosomes or recombinant chromosomes of the invention comprise atelomere.

The invention also provides embodiments wherein any of the Sugarcanemini-chromosomes or recombinant chromosomes of the invention are linearor circular.

In one embodiment, the invention provides for Sugarcane plants or plantcells comprising any of the Sugarcane mini-chromosomes or recombinantchromosomes of the invention. The invention also provides for Sugarcaneplant tissue and Sugarcane seed obtained from the Sugarcane plants ofthe invention.

In another embodiment, the invention provides for Sugarcane plantscomprising any of the Sugarcane mini-chromosomes or recombinantchromosomes of the invention, which may be referred to herein as“adchromosomal” Sugarcane plants. In addition, the invention providesfor Sugarcane plant cells, tissues and seeds obtained from thesemodified plants.

In one embodiment, the invention provides for a Sugarcane plant cellcomprising any of the Sugarcane mini-chromosomes or recombinantchromosomes of the invention that (i) is not integrated into theSugarcane plant cell genome and (ii) confers an altered phenotype on theSugarcane plant cell associated with the expression of at least onestructural gene within the Sugarcane mini-chromosome. The alteredphenotype comprises increased expression of a native gene, decreasedexpression of a native gene, or expression of an exogenous gene. In afurther embodiment, these Sugarcane plant cells also comprise one ormore integrated exogenous structural gene(s).

Another embodiment of the invention is a part of any of the Sugarcaneplants of the invention. Exemplary Sugarcane plant parts of theinvention include a pod, root, sett root, shoot root, root primordial,shoot, primary shoot, secondary shoot, tassle, panicle, arrow, midrib,blade, ligule, auricle, dewlap, blade joint, sheath, node, internode,bud furrow, leaf scar, cutting, tuber, stem, stalk, fruit, berry, nut,flower, leaf, bark, wood, epidermis, vascular tissue, organ, protoplast,crown, callus culture, petiole, petal, sepal, stamen, stigma, style,bud, meristem, cambium, cortex, pith, sheath, silk, ovule or embryo.Other exemplary Sugarcane plant parts are a meiocyte or gamete or ovuleor pollen or endosperm of any of the plants described herein. Otherexemplary plant parts are a seed, seed-piece, embryo, protoplast, cellculture, any group of plant cells organized into a structural andfunctional unit, ratoon, or propagule of any of the Sugarcane plants ofthe invention.

An embodiment of the invention is a progeny of any of the Sugarcaneplants of the invention. These progeny of the invention may be theresult of self-breeding, cross-breeding, apomyxis or clonal propagation.In exemplary embodiments, the invention also provides for progeny thatcomprise a Sugarcane mini-chromosome or recombinant chromosome that isdescended from a parental Sugarcane mini-chromosome or recombinantchromosome that contained a centromere less than about 1000 kilobases inlength, less than about 750 kilobases in length, less than about 600kilobases in length, less than about 500 kilobases in length, less thanabout 400 kilobases in length, less than about 300 kilobases in length,less than about 250 kilobases in length, less than about 200 kilobasesin length, less than about 150 kilobases in length, less than about 100kilobases in length, less than about 90 kilobases in length, less thanabout 85 kilobases in length, less than about 80 kilobases in length,less than about 75 kilobases in length, less than about 70 kilobases inlength, less than about 65 kilobases in length, less than about 60kilobases in length, less than about 55 kilobases in length, less thanabout 50 kilobases in length, less than about 45 kilobases in length,less than about 40 kilobases in length, less than about 35 kilobases inlength, less than about 30 kb in length, less than about 25 kilobases inlength, less than about 20 kb in length, less than about 15 kilobases inlength, less than about 12 kilobases in length, less than about 10 kb inlength, less than about 7 kb in length, less than about 5 kb in length,or less than about 2 kb in length.

In another aspect, the invention provides for methods of making aSugarcane mini-chromosome for use in any of the Sugarcane plants of theinvention. In representative embodiments, these methods compriseidentifying a centromere nucleotide sequence in a Sugarcane genomic DNAlibrary using a multiplicity of diverse probes, and constructing aSugarcane mini-chromosome comprising the centromere nucleotide sequence.These methods may further comprise determining hybridization scores forhybridization of the multiplicity of diverse probes to genomic cloneswithin the Sugarcane genomic nucleic acid library, determining aclassification for genomic clones within the Sugarcane genomic nucleicacid library according to the hybridization scores for at least two ofthe diverse probes, and selecting one or more genomic clones within oneor more classifications for constructing the Sugarcane mini-chromosome.

The invention also contemplates methods of using any of the Sugarcaneplants of the invention to produce a recombinant protein, by growing aSugarcane plant comprising a Sugarcane mini-chromosome or recombinantchromosome that comprises an exogenous nucleic acid encoding the desiredrecombinant protein. Optionally the Sugarcane plant is harvested and thedesired protein product is isolated from the plant. Exemplary proteinproducts include industrial enzymes such as those useful for biofuelproduction.

The invention also contemplates methods of using any of the Sugarcaneplants of the invention to produce a chemical product, by growing aSugarcane plant comprising a Sugarcane mini-chromosome or recombinantchromosome that comprises an exogenous nucleic acid encoding an enzymeinvolved in the synthesis of the chemical product. Optionally theSugarcane plant is harvested and the desired chemical product isisolated from the plant. Exemplary chemical products include sugars,lipids and carbohydrates useful in the production of biofuels.

Another aspect of the invention provides for methods of using any of theSugarcane plants of the invention comprising a Sugarcanemini-chromosomes or recombinant chromosome for a food product, apharmaceutical product or chemical product, according to which asuitable exogenous nucleic acid is expressed in Sugarcane plants orplant cells and the plant or plant cells are grown. The plant maysecrete the product into its growth environment or the product may becontained within the plant, in which case the plant is harvested anddesirable products are extracted.

Thus, the invention contemplates methods of using any of the Sugarcaneplants of the invention comprising a Sugarcane mini-chromosome orrecombinant chromosome to produce a modified food product, for example,by growing a plant that expresses an exogenous nucleic acid that altersthe nutritional content of the plant, and harvesting or processing theSugarcane plant.

The invention also provides for methods of constructing a syntheticarray of repeated nucleotide sequence having Sugarcane centromerefunction comprising the steps of: (a) PCR amplifying a Sugarcanesatellite sequence, (b) cloning the PCR amplified satellite sequenceinto a cloning vector, (c) sequencing the cloned satellite DNA, (d)using a restriction enzyme with an asymmetric recognition sequence toexcise the cloned satellite sequence from the cloning vector, (e)ligating the satellite sequence to one another forming a synthetictandem array, and (f) ligating the synthetic array into a Sugarcanemini-chromosome backbone vector. The invention also provides for anisolated Sugarcane mini-chromosome comprising a synthetic array ofrepeated nucleotide sequence constructed according to the method of theinvention, and Sugarcane plant cells and plants comprising thesemini-chromosomes.

In another embodiment, the invention provides for methods of contactinga Sugarcane cell with a Sugarcane mini-chromosome comprising the stepsof (a) delivering the mini-chromosome to immature differentiated leavesof the apical region of the stem of a Sugarcane plant, wherein themini-chromosome comprises a selectable marker gene, and (b) selectingthe Sugarcane cells expressing the marker gene, wherein expression ofthe marker gene indicates transformation with the mini-chromosome. Theleaves used in this method are immature but are fully differentiated,such as the inner immature leaves of the Sugarcane stem. In an exemplaryembodiment, the mini-chromosome may be delivered by bombarding theimmature leaves with micro-particles comprising the Sugarcanemini-chromosome.

The invention also provides for methods of regenerating a Sugarcaneplant transformed with a Sugarcane mini-chromosome comprising the stepsof (a) obtaining a callus comprising a Sugarcane cell that istransformed by any of the methods of the invention, and (b) growing thecallus in media that may comprise 1%-3% polyvinylpyrrolidone to form aplantlet, wherein the cells of the plantlet are transformed with theSugarcane mini-chromosome. In a further embodiment, the methods ofculturing the callus comprise growing the cells in liquid media for atime period and subsequently culturing the cells in a solid culturemedia. In an exemplary embodiment, the Sugarcane mini-chromosomecomprises a growth regulating gene such as a gene in the auxinbiosynthesis or perception pathways. Such genes may include iaaM (Trpmono-oxygenase), iaaH (Indolo-3-acetamide hydrolase), and ipt (AMPiso-pentenyl transferase). When these three genes are expressed on amini-chromosome(s), IaaMconverts Trp into indole-3-acetamide, which IaaHconverts into auxin. Ipt converts AMP into a cytokinin. The expressionof all three genes allows a cultured cell to grow in the absence ofexogenously supplied hormones.

A further embodiment of the invention is a sugar cane artificialchromosome comprising at least two repeats of a nucleic acid with atleast 98% identity to SEQ ID NO: 204. A still further embodiment of theinvention is plant cell comprising a sugar cane artificial chromosomecomprising at least two repeats of a nucleic acid with at least 98%identity to SEQ ID NO: 204. The plant cell can be a sugar cane plantcell. A further embodiment is a sugar cane artificial chromosomecomprising at least two repeats of a nucleic acid with at least 98%identity to SEQ ID NO: 204 wherein the orientation of the repeats isselected from the group consisting of head to tail, tail to tail, andhead to head. In other embodiments, the percent identity of the nucleicacid to SEQ ID NO: 204 is at least 95%. In still other embodiments, thepercent identity of the nucleic acid to SEQ ID NO: 204 is at least 99%.

Another embodiment of the invention is a sugar cane artificialchromosome comprising at least 15 kbp of a nucleic acid with at least98% identity to SEQ ID NO: 204. A further embodiment of the invention isa plant cell comprising at least 15 kbp of a nucleic acid with at least98% identity to SEQ ID NO: 204. In other embodiments, the percentidentity of the nucleic acid to SEQ ID NO: 204 is at least 95%. In stillother embodiments, the percent identity of the nucleic acid to SEQ IDNO: 204 is at least 99%. The plant cell can be a sugar cane plant cell.

Another embodiment of the invention is a sugar cane artificialchromosome comprising at least 28 kbp of a nucleic acid with at least98% identity to SEQ ID NO: 204. A further embodiment of the invention isa plant cell comprising a sugar cane artificial chromosome comprising atleast 28 kbp of a nucleic acid with at least 98% identity to SEQ ID NO:204. In other embodiments, the percent identity of the nucleic acid toSEQ ID NO: 204 is at least 95%. In still other embodiments, the percentidentity of the nucleic acid to SEQ ID NO: 204 is at least 99%. Theplant cell can be a sugar cane plant cell.

Another embodiment of the invention is a sugar cane artificialchromosome comprising a centromere wherein the centromere comprises atleast two repeats of a nucleic acid with at least 98% identity to SEQ IDNO: 204. A further embodiment of the invention is a plant cellcomprising a sugar cane artificial chromosome comprising a centromerewherein the centromere comprises at least two repeats of a nucleic acidwith at least 98% identity to SEQ ID NO: 204. The plant cell can be asugar cane plant cell. A further embodiment is a sugar cane artificialchromosome comprising a centromere wherein the centromere comprises atleast two repeats of a nucleic acid with at least 98% identity to SEQ IDNO: 204 wherein the orientation of the repeats is selected from thegroup consisting of head to tail, tail to tail and head to head. Inother embodiments, the percent identity of the nucleic acid to SEQ IDNO: 204 is at least 95%. In still other embodiments, the percentidentity of the nucleic acid to SEQ ID NO: 204 is at least 99%.

Another embodiment of the invention is a sugar cane artificialchromosome comprising a centromere wherein the centromere comprises atleast 15 kbp of a nucleic acid with at least 98% identity to SEQ ID NO:204. A further embodiment of the invention is a plant cell comprising acentromere wherein the centromere comprises at least 15 kbp of a nucleicacid with at least 98% identity to SEQ ID NO: 204. The plant cell can bea sugar cane plant cell. In other embodiments, the percent identity ofthe nucleic acid to SEQ ID NO: 204 is at least 95%. In still otherembodiments, the percent identity of the nucleic acid to SEQ ID NO: 204is at least 99%.

Another embodiment of the invention is an isolated nucleic acidcomprising at least 98% identity to SEQ ID NO: 204. A further embodimentof the invention is a plant cell comprising an isolated nucleic acidcomprising at least 98% identity to SEQ ID NO: 204. The plant cell canbe a sugar cane plant cell. In other embodiments, the percent identityof the nucleic acid to SEQ ID NO: 204 is at least 95%. In still otherembodiments, the percent identity of the nucleic acid to SEQ ID NO: 204is at least 99%.

Another embodiment of the invention is an isolated nucleic acidcomprising at least two repeats of a nucleic acid with at least 98%identity to SEQ ID NO: 204. A further embodiment of the invention is aplant cell comprising an isolated nucleic acid comprising at least tworepeats of a nucleic acid with at least 98% identity to SEQ ID NO: 204.The plant cell can be a sugar cane plant cell. A further embodiment isan isolated nucleic acid comprising at least two repeals of a nucleicacid with at least 98% identity to SEQ ID NO: 204 wherein theorientation of the repeats is selected from the group consisting of headto tail, tail to tail and head to head. In other embodiments, thepercent identity of the nucleic acid to SEQ ID NO: 204 is at least 95%.In still other embodiments, the percent identity of the nucleic acid toSEQ ID NO: 204 is at least 99%.

Another embodiment is a method of stably incorporating an autonomouslyreplicating nucleic acid in a sugar cane plant cell comprising the stepsof obtaining a nucleic acid comprising at least two repeats of a nucleicacid with at least 98% identity to SEQ ID NO: 204; transforming a sugarcane plant cell with the nucleic acid; and obtaining a sugar cane plantcell wherein the nucleic acid is autonomously replicating during sugarcane plant cell division. In other embodiments, the percent identity ofthe nucleic acid to SEQ ID NO: 204 is at least 95%. In still otherembodiments, the percent identity of the nucleic acid to SEQ ID NO: 204is at least 99%.

Another embodiment is a method of stably incorporating an autonomouslyreplicating nucleic acid in a sugar cane plant cell comprising the stepsof obtaining a nucleic acid comprising at least 15 kbp of a nucleic acidwith at least 98% identity to SEQ ID NO: 204; transforming a sugar caneplant cell with the nucleic acid; and obtaining a sugar cane plant cellwherein the nucleic acid is autonomously replicating during sugar caneplant cell division. In other embodiments, the percent identity of thenucleic acid to SEQ ID NO: 204 is at least 95%. In still otherembodiments, the percent identity of the nucleic acid to SEQ ID NO: 204is at least 99%.

Another embodiment is a method of stably incorporating an autonomouslyreplicating nucleic acid in a sugar cane plant cell comprising the stepsof obtaining a nucleic acid comprising at least 28 kbp of a nucleic acidwith at least 98% identity to SEQ ID NO: 204; transforming a sugar caneplant cell with the nucleic acid; and obtaining a sugar cane plant cellwherein the nucleic acid is autonomously replicating during sugar caneplant cell division. In other embodiments, the percent identity of thenucleic acid to SEQ ID NO: 204 is at least 95%. In still otherembodiments, the percent identity of the nucleic acid to SEQ ID NO: 204is at least 99%.

Another embodiment is a method of stably incorporating an autonomouslyreplicating nucleic acid in a sugar cane plant cell comprising the stepsof obtaining a nucleic acid comprising a centromere comprising at leasttwo repeats of a nucleic acid with at least 98% identity to SEQ ID NO:204; transforming a sugar cane plant cell with the nucleic acid; andobtaining a sugar cane plant cell wherein the nucleic acid isautonomously replicating during sugar cane plant cell division. In otherembodiments, the percent identity of the nucleic acid to SEQ ID NO: 204is at least 95%. In still other embodiments, the percent identity of thenucleic acid to SEQ ID NO: 204 is at least 99%.

Another embodiment is a method of stably incorporating an autonomouslyreplicating nucleic acid in a sugar cane plant cell comprising the stepsof obtaining a nucleic acid comprising a centromere comprising at least15 kbp of a nucleic acid with at least 98% identity to SEQ ID NO: 204;transforming a sugar cane plant cell with the nucleic acid; andobtaining a sugar cane plant cell wherein the nucleic acid isautonomously replicating during sugar cane plant cell division. In otherembodiments, the percent identity of the nucleic acid to SEQ ID NO: 204is at least 95%. In still other embodiments, the percent identity of thenucleic acid to SEQ ID NO: 204 is at least 99%/o.

Another embodiment is a method of stably incorporating an autonomouslyreplicating nucleic acid in a sugar cane plant cell comprising the stepsof obtaining a nucleic acid comprising a centromere comprising at least28 kbp of a nucleic acid with at least 98% identity to SEQ ID NO: 204;transforming a sugar cane plant cell with the nucleic acid; andobtaining a sugar cane plant cell wherein the nucleic acid isautonomously replicating during sugar cane plant cell division. In otherembodiments, the percent identity of the nucleic acid to SEQ ID NO: 204is at least 95%. In still other embodiments, the percent identity of thenucleic acid to SEQ ID NO: 204 is at least 99%.

Another embodiment is a method of stably incorporating an autonomouslyreplicating nucleic acid in a sugar cane plant cell comprising the stepsof obtaining a nucleic acid comprising at least two repeats of a nucleicacid with at least 98% identity to SEQ ID NO: 204; transforming a sugarcane plant cell with the nucleic acid; and obtaining a sugar cane plantcell wherein the nucleic acid segregates to daughter sugar cane cellsduring sugar cane cell division. In other embodiments, the percentidentity of the nucleic acid to SEQ ID NO: 204 is at least 95%. In stillother embodiments, the percent identity of the nucleic acid to SEQ IDNO: 204 is at least 99%.

Another embodiment is a method of stably incorporating an autonomouslyreplicating nucleic acid in a sugar cane plant cell comprising the stepsof obtaining a nucleic acid comprising at least 15 kbp of a nucleic acidwith at least 98% identity to SEQ ID NO: 204; transforming a sugar caneplant cell with the nucleic acid; and obtaining a sugar cane plant cellwherein the nucleic acid segregates to daughter sugar cane cells duringsugar cane cell division. In other embodiments, the percent identity ofthe nucleic acid to SEQ ID NO: 204 is at least 95%. In still otherembodiments, the percent identity of the nucleic acid to SEQ ID NO: 204is at least 99%.

Another embodiment is a method of stably incorporating an autonomouslyreplicating nucleic acid in a sugar cane plant cell comprising the stepsof obtaining a nucleic acid comprising at least 28 kbp of a nucleic acidwith at least 98% identity to SEQ ID NO: 204; transforming a sugar caneplant cell with the nucleic acid; and obtaining a sugar cane plant cellwherein the nucleic acid segregates to daughter sugar cane cells duringsugar cane cell division. In other embodiments, the percent identity ofthe nucleic acid to SEQ ID NO: 204 is at least 95%. In still otherembodiments, the percent identity of the nucleic acid to SEQ ID NO: 204is at least 99%.

SEQUENCES OF THE INVENTION

The following list indicates the identity of the SEQ ID NOs in thesequence listing:

SEQ ID NOS: 1-201—Sugarcane satellite sequences

SEQ ID NO: 202—Consensus Sugarcane satellite sequence

SEQ ID NO: 203—Sugarcane CRS sequence

SEQ ID NOS: 204—Block of Sugarcane satellite repeat sequence

SEQ ID NOS: 205-221—Primer sequences

SEQ ID NOS: 222-241—Promoter sequences

SEQ ID NOS: 242-251—Primers

DETAILED DESCRIPTION OF THE INVENTION

While this invention is susceptible of embodiment in many differentforms, and will be described herein in detail, specific embodimentsthereof with the understanding that the present disclosure is to beconsidered as an exemplification of the principles of the invention andis not intended to limit the invention to the specific embodimentsillustrated.

The invention provides novel, functional, stable, autonomous Sugarcanemini-chromosomes and recombinant chromosomes comprising centromerescomprising Sugarcane repeat sequences including synthetic sequences.Optionally, the Sugarcane mini-chromosome or recombinant chromosome is“isolated.” The invention also provides for “adchromosomal” Sugarcaneplants described in further detail herein.

One aspect of the invention is related to Sugarcane plants containingfunctional, stable, autonomous Sugarcane mini-chromosomes or recombinantchromosomes, optionally carrying one or more exogenous nucleic acids orcarrying extra copies of a nucleic acid that already exists in theSugarcane genome. Such plants carrying Sugarcane mini-chromosomes orrecombinant chromosomes are contrasted to transgenic plants whose genomehas been altered by integrating exogenous nucleic acid transgenes intothe native Sugarcane chromosomes. In representative embodiments,expression of the exogenous nucleic acid, either constitutively or inresponse to a signal (which may be induced by challenge or a stimulus),or tissue specific expression, or time specific expression, results inan altered phenotype of the plant.

The invention provides for Sugarcane mini-chromosomes or recombinantchromosomes comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 250, 500, 1000 or moreexogenous nucleic acids.

The invention contemplates that Sugarcane plants may be used to carrythe autonomous Sugarcane mini-chromosomes or recombinant chromosomes asdescribed herein. A related aspect of the invention is Sugarcane plantparts or plant tissues, including a pod, root, sett root, shoot root,root primordial, shoot, primary shoot, secondary shoot, tassle, panicle,arrow, midrib, blade, ligule, auricle, dewlap, blade joint, sheath,node, internode, bud furrow, leaf scar, cutting, tuber, stem, stalk,fruit, berry, nut, flower, leaf, bark, wood, epidermis, vascular tissue,organ, protoplast, crown, callus culture, petiole, petal, sepal, stamen,stigma, style, bud, meristem, cambium, cortex, pith, sheath, silk, ovuleor embryo. Other exemplary Sugarcane plant parts are a meiocyte orgamete or ovule or pollen or endosperm of any of the plants of theinvention. Other exemplary plant parts are a seed, seed-piece, embryo,protoplast, cell culture, any group of plant cells organized into astructural and functional unit, ratoon or propagule of any of theSugarcane plants of the invention.

In one embodiment, the exogenous nucleic acid is primarily expressed ina specific location or tissue of a Sugarcane plant, for example, stem,epidermis, vascular tissue, meristem, cambium, cortex, pith, leaf,sheath, flower, root or seed. Tissue-specific expression can beaccomplished with, for example, localized presence of the Sugarcanemini-chromosome or recombinant chromosome, selective maintenance of theSugarcane mini-chromosome or recombinant chromosomes, or with promotersthat drive tissue-specific expression.

Another related aspect of the invention is Sugarcane meiocytes, pollen,ovules, endosperm, seed, somatic embryos, apomyctic embryos, embryosderived from fertilization, vegetative propagules and progeny of theoriginally adchromosomal plant and of its filial generations that retainthe functional, stable, autonomous Sugarcane mini-chromosome orrecombinant chromosome. Such progeny include clonally propagatedSugarcane plants, embryos and plant parts as well as filial progeny fromself- and cross-breeding, and from apomnyxis.

In representative embodiments, the Sugarcane mini-chromosome orrecombinant chromosome is transmitted to subsequent generations ofviable daughter cells during mitotic cell division with a transmissionefficiency of at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99%.

In embodiments of the invention, during meiotic division, the Sugarcanemini-chromosome or recombinant chromosome is transmitted to viablegametes with a transmission efficiency of at least 60%, 70%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% when more than one copy of the Sugarcanemini-chromosome recombinant chromosome is present in the gamete mothercells of the plant. The Sugarcane mini-chromosome or recombinantchromosome can optionally be transmitted to viable gametes duringmeiotic cell division with a transmission frequency of at least 1%, 10%,20%, 30%, 40%, 45%, 46%, 47%, 48%, or 49% when one copy of themini-chromosome or recombinant chromosome is present in the gametemother cells of the Sugarcane plant. According to embodiments of theinvention, for production of seeds via sexual reproduction or byapomyxis the Sugarcane mini-chromosome or recombinant chromosome istransferred into at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99% of viable embryos when cells of the plant contain more than onecopy of the Sugarcane mini-chromosome or recombinant chromosome. Forproduction of seeds via sexual reproduction or by apomyxis fromSugarcane plants with one mini-chromosome or recombinant chromosome percell, the Sugarcane mini-chromosome or recombinant chromosome isoptionally transferred into at least 1%, 10%, 20%, 30%, 40%, 45%, 46%,47%, 48%, or 49% of viable embryos.

In representative embodiments of the invention, a Sugarcanemini-chromosome or recombinant chromosome that comprises an exogenousselectable trait or exogenous selectable marker can be employed toincrease the frequency in subsequent generations of adchromosomal cells,tissues, gametes, embryos, endosperm, seeds, plants or progeny thatcomprise the Sugarcane minichromosome or recombinant chromosome. Inparticular embodiments, the frequency of transmission of Sugarcanemini-chromosomes or recombinant chromosome into viable cells, tissues,gametes, embryos, endosperm, seeds, plants or progeny can be at least95%, 96%, 97%, 98%, 99% or 99.5% after mitosis or meiosis by applying atleast one selection that favors the survival of adchromosomal cells,tissues, gametes, embryos, endosperm, seeds, plants or progeny over suchcells, tissues, gametes, embryos, endosperm, seeds, plants or progenylacking the mini-chromosome or recombinant chromosome.

Transmission efficiency may be measured as the percentage of Sugarcaneprogeny cells or Sugarcane plants that carry the Sugarcanemini-chromosome or recombinant chromosome as measured by one of severalassays taught herein including detection of reporter gene fluorescence,PCR detection of a sequence that is carried by the mini-chromosome orrecombinant chromosome, RT-PCR detection of a gene transcript for a genecarried on the Sugarcane mini-chromosome or recombinant chromosome,Western analysis of a protein produced by a gene carried on theSugarcane mini-chromosome or recombinant chromosome, Southern analysisof the DNA (either in total or a portion thereof) carried by theSugarcane mini-chromosome or recombinant chromosome, fluorescence insitu hybridization (FISH) or in situ localization by repressor binding,to name a few. Any assay used to detect the presence of the Sugarcanemini-chromosome (or a portion of the mini-chromosome) or recombinantchromosome may be used to measure the efficiency that a parental cell orplant transmits the mini-chromosome or recombinant chromosome to itsprogeny. Efficient transmission as measured by some benchmark percentageshould indicate the degree to which the Sugarcane mini-chromosome orrecombinant chromosome is stable through the mitotic and meiotic cycles.

Sugarcane plants of the invention may also contain chromosomallyintegrated exogenous nucleic acid in addition to the autonomousSugarcane mini-chromosomes or recombinant chromosome. The modifiedSugarcane plants or plant parts, including plant tissues of theinvention may include Sugarcane plants that have chromosomal integrationof some portion of the mini-chromosome (e.g. exogenous nucleic acid orcentromere sequences) or recombinant chromosome in some or all cells ofthe plant. In one aspect of the invention, the autonomous Sugarcanemini-chromosome or recombinant chromosome can be isolated fromintegrated exogenous nucleic acid by crossing the modified Sugarcaneplant containing the integrated exogenous nucleic acid with Sugarcaneplants producing some gametes lacking the integrated exogenous nucleicacid and subsequently isolating offspring of the cross, or subsequentcrosses, that are modified but lack the integrated exogenous nucleicacid. This independent segregation of the Sugarcane mini-chromosome orrecombinant chromosome is one measure of the autonomous nature of themini-chromosome.

Another aspect of the invention relates to methods for producing and,optionally, isolating such modified Sugarcane plants containingfunctional, stable, autonomous Sugarcane mini-chromosomes.

In one embodiment, the invention contemplates improved methods forisolating native Sugarcane centromere sequences. In another embodiment,the invention contemplates methods for generating variants of native orartificial Sugarcane centromere sequences by passage through other hostcells such as bacterial or fungal hosts.

In a further embodiment, the invention contemplates methods fordelivering the Sugarcane mini-chromosome into Sugarcane plant cells ortissues to transform the cells or tissues, optionally detectingmini-chromosome presence or assessing mini-chromosome performance, andoptionally generating a Sugarcane plant from such cells or tissues.

Exemplary assays for assessing Sugarcane mini-chromosome or recombinantchromosome performance include lineage-based inheritance assays, use ofchromosome loss agents to demonstrate autonomy, exonuclease digestion,global mitotic mini-chromosome inheritance assays (sectoring assays)with or without the use of agents inducing chromosomal loss, assaysmeasuring expression levels of genes (including marker genes) carried bythe Sugarcane mini-chromosome over time and space in a Sugarcane plant,physical assays for separation of autonomous Sugarcane mini-chromosomesor recombinant chromosomes from endogenous nuclear chromosomes ofSugarcane plants, molecular assays demonstrating conserved Sugarcanemini-chromosome structure or recombinant chromosomes, such as PCR,Southern blots, Sugarcane mini-chromosome rescue, cloning andcharacterization of Sugarcane mini-chromosome sequences present in theSugarcane plant, cytological assays detecting Sugarcane mini-chromosomeor recombinant chromosome presence in the Sugarcane cell's genome (e.g.FISH) and meiotic Sugarcane mini-chromosome or recombinant chromosomeinheritance assays, which measure the levels of mini-chromosome orrecombinant chromosome inheritance into a subsequent generation ofSugarcane plants via meiosis and gametes, embryos, endosperm or seeds.

Another aspect of the invention relates to methods for using Sugarcaneplants containing a Sugarcane mini-chromosome or recombinant chromosomefor producing food products, pharmaceutical products, biofuels andchemical products by appropriate expression of exogenous nucleic acid(s)contained within the mini-chromosome(s) or recombinant chromosome(s).

Yet another aspect of the invention provides novel autonomous Sugarcanemini-chromosomes with novel compositions and structures which are usedto transform plant cells which are in turn used to generate a plant (ormultiple plants). Exemplary Sugarcane mini-chromosomes of the inventionare contemplated to be of a size 2000 kb or less in length. Otherexemplary sizes of Sugarcane mini-chromosomes include less than or equalto, e.g., 1500 kb, 1000 kb, 900 kb, 800 kb, 700 kb, 600 kb, 500 kb, 450kb, 400 kb, 350 kb, 300 kb, 250 kb, 200 kb, 150 kb, 100 kb, 80 kb, 60kb, 40 kb, 35 kb in length. In an exemplary embodiment, themini-chromosome is about 28 kb in length, 42 kb in length, 82 kb inlength, 87 kb in length, 88 kb in length, 97 kb in length, 130 kb inlength, 150 kb in length, 200 kb in length or ranges from 28 kb to 200kb in length.

In a related aspect, novel Sugarcane centromere compositions ascharacterized by sequence content, size or other parameters areprovided. Optionally, the minimal size of Sugarcane centromeric sequenceis utilized in mini-chromosome construction. Exemplary sizes include aSugarcane centromeric nucleic acid segment derived from a portion ofSugarcane genomic DNA or synthesized based on a Sugarcane satelliterepeat sequence, that is less than or equal to 1000 kb, 900 kb, 800 kb,700 kb, 600 kb, 500 kb, 400 kb, 300 kb, 200 kb, 190 kb, 150 kb, 100 kb,95 kb, 90 kb, 85 kb, 80 kb, 75 kb, 70 kb, 65 kb, 60 kb, 55 kb, 50 kb, 45kb, 40 kb, 35 kb, 30 kb, 28 kb, 25 kb, 20 kb, 17 kb, 15 kb, 12 kb, 10kb, 7, kb, 6.4 kb, 5 kb, or 2 kb in length. Exemplary inserts may rangein size from 80 kb to 100 kb, 7 kb to 190 kb, 7 kb to 12 kb, 5 kb to 10kb, 3 kb to 10 kb, 3 kb to 7 kb, 5 kb to 7 kb, 10 to 30 kb, 15 to 30 kb,and 15 to 28 kb. Another related aspect is the novel structure of theSugarcane mini-chromosome, particularly structures lacking bacterialsequences (e.g., sequences required for bacterial propagation), referredto as backbone-free Sugarcane mini-chromosomes.

In other exemplary embodiments, the invention contemplates Sugarcanemini-chromosomes or other vectors comprising centromeric nucleotidesequence that when hybridized to 1, 2, 3, 4, 5, 6, 7, 8 or more of theprobes described in the examples herein, under hybridization conditionsdescribed herein, e.g. low, medium or high stringency, provides relativehybridization scores. Exemplary stringent hybridization conditionscomprise hybridization at 65° C. and washing three times for 15 minuteswith 0.25×SSC, 0.1% SDS at 65° C. Additional exemplary stringenthybridization conditions comprise hybridization in 0.02 M to 0.15 M NaClat temperatures of about 50° C. to 70° C. or 0.5×SSC 0.25% SDS at 65°for 15 minutes, followed by a wash at 65° C. for a half hour orhybridization at 65° C. for 14 hours followed by 3 washings with0.5×SSC, 1% SDS at 65° C. Probe hybridization can be scored visually todetermine a binary (positive versus negative) value, or the probes canbe assigned a score based on the relative strength of theirhybridization on a 10 point scale. For example, relative hybridizationscores of 5 may be used to select clones that hybridize well to theprobe. Alternatively, a hybridization signal greater than background forone or more of these probes can be used to select clones. Modified oradchromosomal Sugarcane plants or plant parts containing such Sugarcanemini-chromosomes are contemplated.

The advantages of the present invention include: provision of anautonomous, independent genetic linkage group for accelerating Sugarcanebreeding; lack of disruption of host Sugarcane genome; multiple gene“stacking” of large and potentially unlimited numbers of genes; uniformgenetic composition of exogenous DNA sequences in plant cells and plantscontaining autonomous Sugarcane mini-chromosomes; defined geneticcontext for predictable gene expression; and higher frequency occurrenceand recovery of Sugarcane plant cells and plants containing stablymaintained exogenous DNA due to elimination of an inefficientintegration step. In addition, Sugarcane mini-chromosomes that increasetotal recoverable sugars or enhance the utility of modified Sugarcaneplants for use in biofuel production are specifically envisioned.

I. Composition of Mini-Chromosomes and Mini-Chromosome Construction

The Sugarcane mini-chromosome vector of the present invention maycontain a variety of elements, including (1) sequences that function asSugarcane centromeres, (2) one or more exogenous nucleic acids,including, for example, plant-expressed genes, or genes for non-codingRNAs, (3) sequences that function as an origin of replication, which maybe included in the region that functions as a plant centromere, (4)optionally, a bacterial plasmid backbone for propagation of the plasmidin bacteria, (5) optionally, sequences that function as plant telomeres,(6) optionally, additional “stuffer DNA” sequences that serve tophysically separate the various components on the Sugarcanemini-chromosome from each other, (7) optionally “buffer” sequences suchas MARs (Matrix Attachment Regions) or SARs (Scaffold AttachmentRegions), (8) optionally marker sequences of any origin, including butnot limited to plant and bacterial origin, (9) optionally, sequencesthat serve as recombination sites, and (10) optionally, “chromatinpackaging sequences” such as cohesion and condensing binding sites.

The Sugarcane mini-chromosomes of the present invention may beconstructed to include various components which are novel, whichinclude, but are not limited to, the Sugarcane centromere comprisingnovel repeating centromeric sequences, as described in further detailbelow.

Novel Centromere Compositions

The centromere in the mini-chromosome of the present invention maycomprise novel repeating Sugarcane centromeric sequences.

Vectors comprising one, two, three, four, five, six, seven, eight, nine,ten, 15 or 20 or more of the elements contained in any of the exemplaryvectors described in the examples below are also contemplated.

The invention specifically contemplates the alternative use of fragmentsor variants (mutants) of any of the nucleic acids described herein thatretain the desired activity, including nucleic acids that function asSugarcane centromeres, nucleic acids that function as promoters or otherregulatory control sequences, or exogenous nucleic acids. Variants mayhave one or more additions, substitutions or deletions of nucleotideswithin the original nucleotide sequence or consensus sequence. Variantsinclude nucleic acid sequences that are at least 50%, 55%, 60, 65, 70,75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100% identical to the original nucleic acid sequence.Variants also include nucleic acid sequences that hybridize under low,medium, high or very high stringency conditions to the original nucleicacid sequence. Similarly, the invention also contemplates thealternative use of fragments or variants of any of the polypeptidesdescribed herein.

The comparison of sequences and determination of percent identitybetween two nucleotide sequences can be accomplished using amathematical algorithm. For example, the percent identity between twonucleotide sequences can be determined using the Needleman and Wunsch(1970) J. Mol. Biol. 48:444-453 algorithm which has been incorporatedinto the GAP program in the GCG software package (available atwww.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix. Theparameters can be set so as to maximize the percent identity.

As used herein, the term “hybridizes under low stringency, mediumstringency, and high stringency conditions” describes conditions forhybridization and washing. Guidance for performing hybridizationreactions can be found in Current Protocols in Molecular Biology (1989)John Wiley & Sons, N.Y., 6.3.1-6.3.6, which is incorporated byreference. Aqueous and non-aqueous methods are described in thatreference and either can be used. Specific hybridization conditionsreferred to herein are as follows: 1) low stringency hybridizationconditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by two washes in 0.5×SSC, 0.1% SDS, at least at 50° C.; 2)medium stringency hybridization conditions in 6×SSC at about 45° C.,followed by one or more washes in 0.2×SSC, 0.1% SDS at 55° C.; 3) highstringency hybridization conditions are hybridization at 65° C. for12-18 hours and washing three times for 15-90 minutes with 0.25×SSC,0.1% SDS at 65° C. Additional exemplary stringent hybridizationconditions comprise 6×SSC at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 65° C. Other exemplary highly selectiveor stringent hybridization conditions comprise 0.02 M to 0.15 M NaCl attemperatures of about 50° C. to 70° C. or 0.5×SSC 0.25% SDS at 65° for12-15 hours, followed three washes at 65° C. for 15-90 minutes each.

Sugarcane Mini-Chromosome Sequence Content and Structure

Sugarcane-expressed genes from non-plant sources may be modified toaccommodate Sugarcane codon usage, to insert preferred motifs near thetranslation initiation ATG codon, to remove sequences recognized inplants as 5′ or 3′ splice sites, or to better reflect plant GC/ATcontent. Plant genes typically have a GC content of more than 35%, andcoding sequences which are rich in A and T nucleotides can beproblematic. For example, ATTTA motifs may destabilize mRNA; plantpolyadenylation signals such as AATAAA at inappropriate positions withinthe message may cause premature truncation of transcription; andmonocotyledons such as Sugarcane may recognize AT-rich sequences assplice sites.

Each exogenous nucleic acid or Sugarcane-expressed gene may include apromoter, a coding region and a terminator sequence, which may beseparated from each other by restriction endonuclease sites orrecombination sites or both. Genes may also include introns, which maybe present in any number and at any position within the transcribedportion of the gene, including the 5′ untranslated sequence, the codingregion and the 3′ untranslated sequence. Introns may be natural plantintrons derived from any plant, or artificial introns based on thesplice site consensus that has been defined for plant species. Someintron sequences have been shown to enhance expression in plants.Optionally the exogenous nucleic acid may include a planttranscriptional terminator, non-translated leader sequences derived fromviruses that enhance expression, a minimal promoter, or a signalsequence controlling the targeting of gene products to plantcompartments or organelles.

The coding regions of the genes can encode any protein, including butnot limited to visible marker genes (for example, fluorescent proteingenes, other genes conferring a visible phenotype to the plant) or otherscreenable or selectable marker genes (for example, conferringresistance to antibiotics, herbicides or other toxic compounds orencoding a protein that confers a growth advantage to the cellexpressing the protein) or genes which confer some commercial oragronomic value to the modified or adchromosomal Sugarcane plant.Multiple genes can be placed on the same Sugarcane mini-chromosomevector. The genes may be separated from each other by restrictionendonuclease sites, homing endonuclease sites, recombination sites orany combinations thereof. Alternatively, the cloning process can beexecuted in a manner that destroys the intervening restriction sites.Any number of genes can be present.

The Sugarcane mini-chromosome vector may also contain a bacterialplasmid backbone for propagation of the plasmid in bacteria such as E.coli, A. tumefaciens, or A. rhizogenes. The plasmid backbone may be thatof a low-copy vector or in other embodiments it may be desirable to usea mid to high level copy backbone. In one embodiment of the invention,this backbone contains the replicon of the F′ plasmid of E. coli.However, other plasmid replicons, such as the bacteriophage P1 replicon,or other low-copy plasmid systems such as the RK2 replication origin,may also be used. The backbone may include one or severalantibiotic-resistance genes conferring resistance to a specificantibiotic to the bacterial cell in which the plasmid is present.Bacterial antibiotic-resistance genes include but are not limited tokanamycin-, ampicillin-, chloramphenicol-, streptomycin-,spectinomycin-, tetracycline- and gentamycin-resistance genes.

The Sugarcane mini-chromosome vector may also contain plant telomeres.An exemplary telomere sequence is TTTAGGG or its complement. Telomeresare specialized DNA structures at the ends of linear chromosomes thatfunction to stabilize the ends and facilitate the complete replicationof the extreme termini of the DNA molecule (Richards et al., Cell, 1988Apr. 8; 53(1):127-36; Ausubel et al., Current Protocols in MolecularBiology, Wiley & Sons, 1997).

Additionally, the Sugarcane mini-chromosome vector may contain “stufferDNA” sequences that serve to separate the various components on themini-chromosome (centromere, genes, telomeres) from each other. Thestuffer DNA may be of any origin, prokaryotic or eukaryotic, and fromany genome or species, plant, animal, microbe or organelle, or may be ofsynthetic origin. The stuffer DNA can range from 100 bp to 10 Mb inlength and can be repetitive in sequence, with unit repeats from 10 to1,000,000 bp. Examples of repetitive sequences that can be used asstuffer DNAs include but are not limited to: rDNA, satellite repeats,retroelements, transposons, pseudogenes, transcribed genes,microsatellites, tDNA genes, short sequence repeats and combinationsthereof. Alternatively, the stuffer DNA can consist of unique,non-repetitive DNA of any origin or sequence. The stuffer sequences mayalso include DNA with the ability to form boundary domains, such as butnot limited to scaffold attachment regions (SARs) or matrix attachmentregions (MARs). The stuffer DNA may be entirely synthetic, composed ofrandom sequence. In this case, the stuffer DNA may have any basecomposition, or any A/T or G/C content. For example, the G/C content ofthe stuffer DNA could resemble that of the plant (˜30-40%), or could bemuch lower (0-30%) or much higher (40-100%). Alternatively, the stuffersequences could be synthesized to contain an excess of any givennucleotide such as A, C, G or T. Different synthetic stuffers ofdifferent compositions may also be combined with each other. For examplea fragment with low G/C content may be flanked or abutted by a fragmentof medium or high G/C content, or vice versa.

In one embodiment of the invention, the Sugarcane mini-chromosome has acircular structure without telomeres. In another embodiment, theSugarcane mini-chromosome has a circular structure with telomeres. In athird embodiment, the Sugarcane mini-chromosome has a linear structurewith telomeres, for example, as would result if a “linear” structurewere to be cut with a unique endonuclease, exposing the telomeres at theends of a DNA molecule that contains all of the sequence contained inthe original, closed construct with the exception of anantibiotic-resistance gene. In a fourth embodiment of the invention, thetelomeres could be placed in such a manner that the bacterial replicon,backbone sequences, antibiotic-resistance genes and any other sequencesof bacterial origin and present for the purposes of propagation of theSugarcane mini-chromosome in bacteria, can be removed from theplant-expressed genes, the centromere, telomeres, and other sequences bycutting the structure with, for example, a unique endonuclease. Thisresults in a Sugarcane mini-chromosome from which much of, or even all,bacterial sequences have been removed. In this embodiment, bacterialsequence present between or among the plant-expressed genes or otherSugarcane mini-chromosome sequences would be excised prior to removal ofthe remaining bacterial sequences by cutting the Sugarcanemini-chromosome with an endonuclease and re-ligating the structure suchthat the antibiotic-resistance gene has been lost. The uniqueendonuclease site may be the recognition sequence of any of a number ofendonucleases including but not limited to restriction endonucleases,meganucleases, or homing endonuclease. Alternatively, the endonucleasesand their sites can be replaced with any specific DNA cutting mechanismand its specific recognition site such as rare-cutting endonuclease orrecombinase and its specific recognition site, as long as that site ispresent in the Sugarcane mini-chromosomes only at the indicatedpositions.

Various structural configurations are possible by which Sugarcanemini-chromosome elements can be oriented with respect to each other. ASugarcane centromere can be placed on a Sugarcane mini-chromosome eitherbetween genes or outside a cluster of genes next to one telomere or nextto the other telomere. Stuffer DNAs can be combined with theseconfigurations to place the stuffer sequences inside the telomeres,around the centromere between genes or any combination thereof. Thus, alarge number of alternative Sugarcane mini-chromosome structures arepossible, depending on the relative placement of centromere DNA, genes,stuffer DNAs, bacterial sequences, telomeres, and other sequences. Thesequence content of each of these variants is the same, but theirstructure may be different depending on how the sequences are placed.These variations in architecture are possible both for linear and forcircular Sugarcane mini-chromosomes.

Exemplary Centromere Components

Sugarcane centromere components may be isolated or derived from a nativeplant genome, for example, modified through recombinant techniques orthrough the cell-based techniques described below. Alternatively, whollyartificial centromere components may be constructed using as a generalguide the sequence of native Sugarcane centromeres such as nativesatellite repeat sequences. Combinations of centromere componentsderived from natural sources and/or combinations of naturally derivedand artificial components are also contemplated.

In one embodiment, the Sugarcane centromere contains n copies of arepeated nucleotide sequence obtained by the methods disclosed herein;wherein n is at least 2. In another embodiment, the Sugarcane centromerecontains n copies of interdigitated repeats. An interdigitated repeat isa DNA sequence that consists of two distinct repetitive elements thatcombine to create a unique permutation. Potentially any number of repeatcopies capable of physically being placed on the recombinant constructcould be included on the construct, including about 5, 10, 15, 20, 30,50, 75, 100, 150, 200, 300, 400, 500, 750, 1,000, 1,500, 2,000, 3,000,5,000, 7,500, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000,80,000, 90,000 and about 100,000, including all ranges in-between suchcopy numbers. Moreover, the copies, while largely identical, can varyfrom each other. Such repeat variation is commonly observed in naturallyoccurring centromeres. The length of the repeat may vary, and can rangefrom about 20 bp to about 360 bp, from about 20 bp to about 250 bp, fromabout 50 bp to about 225 bp, from 20 bp to 137 bp, from about 75 bp toabout 210 bp, from about 100 bp to about 205 bp, from about 125 bp toabout 200 bp, from about 150 bp to about 195 bp, from about 160 bp toabout 190 and from about 170 bp to about 185 bp including about 180 bp.Exemplary repeats include without limitation a 92 bp repeat, a 97 bprepeat or a 100 bp repeat. Larger repeats including those up to 3,465 bpor 3,500 bp or 3,600 bp or 3,700 bp are also anticipated by the currentinvention.

The invention contemplates that two or more of these Sugarcane repeatednucleotide sequences, or similar Sugarcane repeated nucleotidesequences, may be oriented head to tail within the centromere. The term“head to tail” refers to multiple consecutive copies of the same orsimilar repeated nucleotide sequence (e.g., at least 70% identical) thatare in the same 5′-3′ orientation. The invention also contemplates thattwo or more of these repeated nucleotide sequences may be consecutivewithin the Sugarcane centromere. The term “consecutive” refers to thesame or similar repeated nucleotide sequences (e.g., at least 70%identical) that follow one after another without being interrupted byother significant sequence elements. Such consecutive repeatednucleotide sequences may be in any orientation, e.g. head to tail, tailto tail, or head to head, and may be separated by n number ofnucleotides, wherein n ranges from 1 to 10, or 1 to 20, or 1 to 30, or 1to 40, or 1 to 50. Exemplary repeated nucleotide sequences derived fromSugarcane, and identified by the methods described herein, are set outas SEQ ID NOS: 1-202, SEQ ID NO: 204 and CRS (SEQ ID NO: 203).

Modification of Sugarcane Centromeres Isolated from Native Plant Genome

Modification and changes may be made in the Sugarcane centromeric DNAsegments specifically described herein and still obtain a functionalmolecule with desirable characteristics. Such modified Sugarcanecentromeres are also encompassed by the present invention. The followingis a discussion based upon changing the nucleic acids of a Sugarcanecentromere to create an equivalent, or even an improved, secondgeneration molecule.

In particular embodiments of the invention, mutated Sugarcanecentromeric sequences are contemplated to be useful for increasing theutility of the Sugarcane centromere. Without being bound by any theoryof the invention, it is specifically contemplated that the function ofthe Sugarcane centromeres of the current invention may be based in partor in whole upon the secondary structure of the DNA sequences of theSugarcane centromere, modification of the DNA with methyl groups orother adducts, and/or the proteins which interact with the Sugarcanecentromere. By changing the DNA sequence of the Sugarcane centromere,one may alter the affinity of one or more centromere-associatedprotein(s) for the Sugarcane centromere and/or the secondary structureor modification of the Sugarcane centromeric sequences, thereby changingthe activity of the Sugarcane centromere. Alternatively, changes may bemade in the Sugarcane centromeres of the invention which do not affectthe activity of the Sugarcane centromere. Changes in the Sugarcanecentromeric sequences which reduce the size of the DNA segment needed toconfer Sugarcane centromere activity are contemplated to be particularlyuseful in the current invention, as would changes which increased thefidelity with which the Sugarcane centromere was transmitted duringmitosis or meiosis.

Modification of Sugarcane Centromeres by Passage Through BacteriaSugarcane or Other Hosts or Processes

In the methods of the present invention, the resulting Sugarcanemini-chromosome DNA sequence may also be a derivative of the parentalclone or Sugarcane centromere clone having substitutions, deletions,insertions, duplications and/or rearrangements of one or morenucleotides in the nucleic acid sequence. Such nucleotide mutations mayoccur individually or consecutively in stretches of about 1, 2, 3, 4, 5,6, 7, 8, 9 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125,150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000,2000, 4000, 8000, 10000, 50000, 100000, and about 200000, including allranges in-between.

Variations of Sugarcane mini-chromosomes may arise through passage ofSugarcane mini-chromosomes through various hosts including virus,bacteria, yeast, or another prokaryotic or eukaryotic organism and mayoccur through passage of multiple hosts or an individual host.Variations may also occur by replicating the Sugarcane mini-chromosomein vitro.

Derivatives may be identified through sequence analysis, or variationsin Sugarcane mini-chromosome molecular weight through electrophoresissuch as, but not limited to, CHEF gel analysis, column or gradientseparation, or any other methods used in the field to determine and/oranalyze DNA molecular weight or sequence content. Alternately,derivatives may be identified by the altered activity of a derivative inconferring Sugarcane centromere function to a Sugarcane mini-chromosome.

Production or Synthesis of Synthetic Sugarcane Centromere RepeatSequences

These artificially synthesized repeated nucleotide sequences of theinvention may be derived from natural Sugarcane centromere sequences,combinations or fragments of natural Sugarcane centromere sequencesincluding a combination of repeats of different lengths, a combinationof different sequences, a combination of both different repeat lengthsand different sequences, a combination of different artificiallysynthesized sequences or a combination of natural Sugarcane centromeresequence(s) and artificially synthesized sequence(s). The syntheticnucleotide sequences and arrays of these synthetic repeat sequences maybe generated using any technique known in the art including PCR fromgenomic DNA, e.g. the methods described in Example 1, or by custompolynucleotide synthesis.

Polynucleotide synthesis is the non-biological, chemical synthesis ofdefined sequences of nucleic acids using automated synthesizers.Oligonucleotides may be chemically synthesized, purified and then theseoligonucleotides are connected by specific annealing and standardligation or polymerase reactions. Exemplary ligation methods includeligation of phosphorylated overlapping oligonucleotides (Gupta et al.Proc. Natl Acad. Sci. USA, 60, 1338-1344, Fuhrmann et al. Plant J. 1999August; 19(3):353-61), the FokI method (Mandecki et al. Gene, 68,101-107) and a modified form of ligase chain reaction for genesynthesis. In addition, PCR assembly approaches may be used whichgenerally employ oligonucleotides of 40-50 nt long that overlap eachother. These oligonucleotides are designed to cover most of the sequenceof both strands, and the full-length molecule is generated progressivelyby overlap extension PCR (Stemmer et al. Gene, 164, 49-53).,thermodynamically balanced inside-out PCR (Gao et al. Nucleic Acids Res.2003 Nov. 15; 31(22):e143) or combined approaches (Young et al. NucleicAcids Res. 2004 Apr. 15; 32(7):e59).

Exemplary Exogenous Nucleic Acids Including Plant-Expressed Genes

Of particular interest in the present invention are exogenous nucleicacids which when introduced into Sugarcane plants will alter thephenotype of the plant, a plant organ, plant tissue, or a portion of theplant. Exemplary exogenous nucleic acids encode polypeptides. Otherexemplary exogenous nucleic acids alter expression of exogenous orendogenous genes, either increasing or decreasing expression, optionallyin response to a specific signal or stimulus.

As used herein, the term “trait” can refer either to the alteredphenotype of interest or the nucleic acid which causes the alteredphenotype of interest.

One of the major purposes of transformation of Sugarcane is to add somecommercially desirable, agronomically important traits to the plant.Such traits include, but are not limited to, enhanced production oftotal recoverable sugars; utility for production of biofuels; herbicideresistance or tolerance, insect (pest) resistance or tolerance; diseaseresistance or tolerance (viral, bacterial, fungal, nematode or otherpathogens); stress tolerance and/or resistance, as exemplified byresistance or tolerance to drought, heat, chilling, freezing, excessivemoisture, salt stress, mechanical stress, extreme acidity, alkalinity,toxins, UV light, ionizing radiation or oxidative stress; increasedyields, increased biomass, whether in quantity or quality; enhanced oraltered nutrient acquisition and enhanced or altered metabolicefficiency; enhanced or altered nutritional content and makeup of planttissues used for food, feed, fiber or processing; physical appearance;male sterility; drydown; standability; prolificacy; starch quantity andquality; oil quantity and quality; protein quality and quantity; aminoacid composition; modified chemical production; altered pharmaceuticalor nutraceutical properties; altered bioremediation properties;increased biomass; altered growth rate; altered fitness; alteredbiodegradability; altered CO₂ fixation; presence of bioindicatoractivity; altered digestibility by humans or animals; alteredallergenicity; altered mating characteristics; altered pollen dispersal;improved environmental impact; altered nitrogen fixation capability; theproduction of a pharmaceutically active protein; the production of asmall molecule with medicinal properties; the production of a chemicalincluding those with industrial utility; the production ofnutraceuticals, food additives, carbohydrates, RNAs, lipids, fuels,dyes, pigments, vitamins, scents, flavors, vaccines, antibodies,hormones, and the like; and alterations in plant architecture ordevelopment, including changes in developmental timing, photosynthesis,signal transduction, cell growth, reproduction, or differentiation.Additionally one could create a library of an entire genome (or aportion thereof) from any organism or organelle including mammals,plants, microbes, fungi, or bacteria, represented on Sugarcanemini-chromosomes.

In one embodiment, the Sugarcane plant comprising a Sugarcanemini-chromosome or recombinant chromosome may exhibit increased ordecreased expression or accumulation of a product of the plant, whichmay be a natural product of the plant or a new or altered product of theplant. Exemplary products include an enzyme, an RNA molecule, anutritional protein, a structural protein, an amino acid, a lipid, afatty acid, a polysaccharide, a sugar, an alcohol, an alkaloid, acarotenoid, a propanoid, a phenylpropanoid, or terpenoid, a steroid, aflavonoid, a phenolic compound, an anthocyanin, a pigment, a vitamin ora plant hormone. In another embodiment, the Sugarcane plant comprising aSugarcane mini-chromosome or recombinant chromosome has enhanced ordiminished requirements for light, water, nitrogen, or trace elements.In another embodiment the Sugarcane plant comprising a Sugarcanemini-chromosome or recombinant chromosome has an enhanced ability tocapture or fix nitrogen from its environment. In yet another embodiment,the Sugarcane plant comprising a Sugarcane mini-chromosome orrecombinant chromosome is enriched for an essential amino acid as aproportion of a protein fraction of the plant. The protein fraction maybe, for example, total seed protein, soluble protein, insoluble protein,water-extractable protein, and lipid-associated protein. The Sugarcanemini-chromosome or recombinant chromosome may include genes that causethe overexpression, underexpression, antisense modulation, sensesuppression, inducible expression, inducible repression, or induciblemodulation of another gene.

A brief summary of exemplary improved properties and polypeptides ofinterest for either increased or decreased expression is provided below.

(1) Herbicide Resistance

An herbicide resistance (or tolerance) trait is a characteristic of aSugarcane plant comprising a Sugarcane mini-chromosome or recombinantchromosome that is resistant to dosages of an herbicide that istypically lethal to a wild type plant. Exemplary herbicides for whichresistance is useful in a plant include glyphosate herbicides,phosphinothricin herbicides, oxynil herbicides, imidazolinoneherbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylureaherbicides, bialaphos herbicides, sulfonamide herbicides and glufosinateherbicides. Other herbicides would be useful as would combinations ofherbicide genes on the same Sugarcane mini-chromosome or recombinantchromosome.

The genes encoding phosphinothricin acetyltransferase (bar), glyphosatetolerant EPSP synthase genes, glyphosate acetyltransferase, theglyphosate degradative enzyme gene gox encoding glyphosateoxidoreductase, deh (encoding a dehalogenase enzyme that inactivatesdalapon), herbicide resistant (e.g., sulfonylurca and imidazolinone)acetolactate synthase, and bxn genes (encoding a nitrilase enzyme thatdegrades bromoxynil) are good examples of herbicide resistant genes foruse in transformation. The bar gene codes for an enzyme,phosphinothricin acetyltransferase (PAT), which inactivates theherbicide phosphinothricin and prevents this compound from inhibitingglutamine synthetase enzymes. The enzyme 5 enolpyruvylshikimate 3phosphate synthase (EPSP Synthase), is normally inhibited by theherbicide N (phosphonomethyl)glycine (glyphosate). However, genes areknown that encode glyphosate resistant EPSP synthase enzymes. Thesegenes are particularly contemplated for use in plant transformation. Thedeh gene encodes the enzyme dalapon dehalogenase and confers resistanceto the herbicide dalapon. The bxn gene codes for a specific nitrilaseenzyme that converts bromoxynil to a non herbicidal degradation product.The glyphosate acetyl transferase gene inactivates the herbicideglyphosate and prevents this compound from inhibiting EPSP synthase.

Polypeptides that may produce plants having tolerance to plantherbicides include polypeptides involved in the shikimate pathway, whichare of interest for providing glyphosate tolerant plants. Suchpolypeptides include polypeptides involved in biosynthesis ofchorismate, phenylalanine, tyrosine and tryptophan.

(ii) Insect Resistance

Potential insect resistance (or tolerance) genes that can be introducedinclude Bacillus thuringiensis toxin genes or Bt genes (Watrud et al.,In: Engineered Organisms and the Environment, 1985). Bt genes mayprovide resistance to lepidopteran or coleopteran pests such as EuropeanCorn Borer (ECB). Exemplary Bt toxin genes for use in such embodimentsinclude the CryIA(b) and CryIA(c) genes. Endotoxin genes from otherspecies of B. thuringiensis which affect insect growth or developmentalso may be employed in this regard.

It is contemplated that in some embodiments Bt genes for use in theSugarcane mini-chromosomes or recombinant chromosomes disclosed hereinwill be those in which the coding sequence has been modified to effectincreased expression in plants, and for example, in monocot plantsincluding Sugarcane. Means for preparing synthetic genes are well knownin the art and are disclosed in, for example, U.S. Pat. No. 5,500,365and U.S. Pat. No. 5,689,052, each of the disclosures of which arespecifically incorporated herein by reference in their entirety.Examples of such modified Bt toxin genes include a synthetic Bt CryIA(b)gene (Perlak et al., Proc. Natl. Acad. Sci. USA, 88:3324-3328, 1991),and the synthetic CryIA(c) gene termed 1800b (PCT Application WO95/06128). Some examples of other Bt toxin genes known to those of skillin the art are given in Table 1 below.

TABLE 1 Bacillus thuringiensis Endotoxin Genes^(a) New Nomenclature OldNomenclature GenBank Accession Cry1Aa CryIA(a) M11250 Cry1Ab CryIA(b)M13898 Cry1Ac CryIA(c) M11068 Cry1Ad CryIA(d) M73250 Cry1Ae CryIA(e)M65252 Cry1Ba CryIB X06711 Cry1Bb ET5 L32020 Cry1Bc PEG5 Z46442 Cry1BdCryE1 U70726 Cry1Ca CryIC X07518 Cry1Cb CryIC(b) M97880 Cry1Da CryIDX54160 Cry1Db PrtB Z22511 Cry1Ea CryIE X53985 Cry1Eb CryIE(b) M73253Cry1Fa CryIF M63897 Cry1Fb PrtD Z22512 Cry1Ga PrtA Z22510 Cry1Gb CryH2U70725 Cry1Ha PrtC Z22513 Cry1Hb U35780 Cry1Ia CryV X62821 Cry1Ib CryVU07642 Cry1Ja ET4 L32019 Cry1Jb ET1 U31527 Cry1K U28801 Cry2Aa CryIIAM31738 Cry2Ab CryIIB M23724 Cry2Ac CryIIC X57252 Cry3A CryIIIA M22472Cry3Ba CryIIIB X17123 Cry3Bb CryIIIB2 M89794 Cry3C CryIIID X59797 Cry4ACryIVA Y00423 Cry4B CryIVB X07423 Cry5Aa CryVA(a) L07025 Cry5Ab CryVA(b)L07026 Cry6A CryVIA L07022 Cry6B CryVIB L07024 Cry7Aa CryIIIC M64478Cry7Ab CryIIICb U04367 Cry8A CryIIIE U04364 Cry8B CryIIIG U04365 Cry8CCryIIIF U04366 Cry9A CryIG X58120 Cry9B CryIX X75019 Cry9C CryIH Z37527Cry10A CryIVC M12662 Cry11A CryIVD M31737 Cry11B Jeg80 X86902 Cry12ACryVB L07027 Cry13A CryVC L07023 Cry14A CryVD U13955 Cry15A 34 kDaM76442 Cry16A cbm7l X94146 Cry17A cbm71 X99478 Cry18A CryBP1 X99049Cry19A Jeg65 Y08920 Cyt1Aa CytA X03182 Cyt1Ab CytM X98793 Cyt2A CytBZ14147 Cyt2B CytB U52043 ^(a)Adapted from:http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html

Protease inhibitors also may provide insect resistance (Johnson et al.,Proc Natl Acad Sci USA. 1989 December; 86(24): 9871-9875), and will thushave utility in Sugarcane transformation. The use of a pinII gene incombination with a Bt toxin gene, the combined effect of which has beendiscovered to produce synergistic insecticidal activity is envisioned tobe particularly useful. Other genes which encode inhibitors of theinsect's digestive system, or those that encode enzymes or co factorsthat facilitate the production of inhibitors, also may be useful. Thisgroup may be exemplified by oryzacystatin and amylase inhibitors such asthose from wheat and barley.

Amylase inhibitors are found in various plant species and are used toward off insect predation via inhibition of the digestive amylases ofattacking insects. Several amylase inhibitor genes have been isolatedfrom plants and some have been introduced as exogenous nucleic acids,conferring an insect resistant phenotype that is potentially useful(“Plants, Genes, and Crop Biotechnology” by Maarten J. Chrispeels andDavid E. Sadava (2003) Jones and Bartlett Press).

Genes encoding lectins may confer additional or alternative insecticideproperties. Lectins are multivalent carbohydrate binding proteins whichhave the ability to agglutinate red blood cells from a range of species.Lectins have been identified recently as insecticidal agents withactivity against weevils, ECB and rootworm (Murdock et al.,Phytochemistry, 29:85-89, 1990, Czapla & Lang, J. Econ. Entomol.,83:2480-2485, 1990). Lectin genes contemplated to be useful include, forexample, barley and wheat germ agglutinin (WGA) and rice lectins(Gatehouse et al., J. Sci. Food. Agric., 35:373-380, 1984).

Genes controlling the production of large or small polypeptides activeagainst insects when introduced into the insect pests, such as, e.g.,lytic peptides, peptide hormones and toxins and venoms, form anotheraspect of the invention. For example, it is contemplated that theexpression of juvenile hormone esterase, directed towards specificinsect pests, also may result in insecticidal activity, or perhaps causecessation of metamorphosis (Hammock et al., Nature, 344:458-461, 1990).

Genes that encode enzymes that affect the integrity of the insectcuticle form yet another aspect of the invention. Such genes includethose encoding, e.g., chitinase, proteases, lipases and also genes forthe production of nikkomycin, a compound that inhibits chitin synthesis,the introduction of any of which is contemplated to produce insectresistant plants. Genes that code for activities that affect insectmolting, such as those affecting the production of ecdysteroid UDPglucosyl transferase, also fall within the scope of the useful exogenousnucleic acids of the present invention.

Genes that code for enzymes that facilitate the production of compoundsthat reduce the nutritional quality of the host Sugarcane plant toinsect pests also are encompassed by the present invention. It may bepossible, for instance, to confer insecticidal activity on a Sugarcaneplant by altering its sterol composition. Sterols are obtained byinsects from their diet and are used for hormone synthesis and membranestability. Therefore alterations in plant sterol composition byexpression of novel genes, e.g., those that directly promote theproduction of undesirable sterols or those that convert desirablesterols into undesirable forms, could have a negative effect on insectgrowth and/or development and hence endow the plant with insecticidalactivity. Lipoxygenases are naturally occurring plant enzymes that havebeen shown to exhibit anti nutritional effects on insects and to reducethe nutritional quality of their diet. Therefore, further embodiments ofthe invention concern modified plants with enhanced lipoxygenaseactivity which may be resistant to insect feeding.

Tripsacum dactyloides is a species of grass that is resistant to certaininsects, including root worm. It is anticipated that genes encodingproteins that are toxic to insects or are involved in the biosynthesisof compounds toxic to insects will be isolated from Tripsacum and thatthese novel genes will be useful in conferring resistance to insects. Itis known that the basis of insect resistance in Tripsacum is genetic,because said resistance has been transferred to Zea mays via sexualcrosses (Branson and Guss, Proceedings North Central BranchEntomological Society of America, 27:91-95, 1972). It is furtheranticipated that other cereal, monocot or dicot plant species may havegenes encoding proteins that are toxic to insects which would be usefulfor producing insect resistant Sugarcane plants.

Further genes encoding proteins characterized as having potentialinsecticidal activity also may be used as exogenous nucleic acids inaccordance herewith. Such genes include, for example, the cowpea trypsininhibitor (CpTI; Hilder et al., Nature, 330:160-163, 1987) which may beused as a rootworm deterrent; genes encoding avermectin (Avermectin andAbamectin., Campbell, W. C., Ed., 1989; Ikeda et al., J. Bacteriol.,169:5615-5621, 1987) which may prove particularly useful as a cornrootworm deterrent; ribosome inactivating protein genes; and even genesthat regulate plant structures. Sugarcane plants comprising a Sugarcanemini-chromosome or recombinant chromosome comprising anti insectantibody genes and genes that code for enzymes that can convert a nontoxic insecticide (pro insecticide) applied to the outside of the plantinto an insecticide inside the plant also are contemplated.

Polypeptides that may improve Sugarcane tolerance to the effects ofplant pests or pathogens include proteases, polypeptides involved inanthocyanin biosynthesis, polypeptides involved in cell wall metabolism,including cellulases, glucosidases, pectin methylesterase, pectinase,polygalacturonase, chitinase, chitosanase, and cellulose synthase, andpolypeptides involved in biosynthesis of terpenoids or indole forproduction of bioactive metabolites to provide defense againstherbivorous insects. It is also anticipated that combinations ofdifferent insect resistance genes on the same Sugarcane mini-chromosomeor recombinant chromosome will be particularly useful.

Vegetative Insecticidal Proteins (VIP) are another class of proteinsoriginally found to be produced in the vegetative growth phase of thebacterium, Bacillus cereus, but do have a spectrum of insect lethalitysimilar to the insecticidal genes found in strains of Bacillusthuringiensis. Both the vip1a and vip3A genes have been isolated andhave demonstrated insect toxicity. It is anticipated that such genes maybe used in modified plants to confer insect resistance (“Plants, Genes,and Crop Biotechnology” by Maarten J. Chrispeels and David E. Sadava(2003) Jones and Bartlett Press).

(ii) Environment or Stress Resistance

Improvement of a Sugarcane plant's ability to tolerate variousenvironmental stresses such as, but not limited to, drought, excessmoisture, chilling, freezing, high temperature, salt, and oxidativestress, also can be effected through expression of novel genes. It isproposed that benefits may be realized in terms of increased resistanceto freezing temperatures through the introduction of an “antifreeze”protein such as that of the Winter Flounder (Cutler et al., J. PlantPhysiol., 135:351-354, 1989) or synthetic gene derivatives thereof.Improved chilling tolerance also may be conferred through increasedexpression of glycerol 3 phosphate acetyltransferase in chloroplasts(Wolter et al., The EMBO J., 4685-4692, 1992). Resistance to oxidativestress (often exacerbated by conditions such as chilling temperatures incombination with high light intensities) can be conferred by expressionof superoxide dismutase (Gupta et al., 1993), and may be improved byglutathione reductase (Bowler et al., Ann Rev. Plant Physiol.,43:83-116, 1992). Such strategies may allow for tolerance to freezing innewly emerged fields as well as extending later maturity higher yieldingvarieties to earlier relative maturity zones.

It is contemplated that the expression of novel genes that favorablyaffect Sugarcane plant water content, total water potential, osmoticpotential, or turgor will enhance the ability of the plant to toleratedrought. As used herein, the terms “drought resistance” and “droughttolerance” are used to refer to a Sugarcane plant's increased resistanceor tolerance to stress induced by a reduction in water availability, ascompared to normal circumstances, and the ability of the plant tofunction and survive in lower water environments. In this aspect of theinvention it is proposed, for example, that the expression of genesencoding for the biosynthesis of osmotically active solutes, such aspolyol compounds, may impart protection against drought. Within thisclass are genes encoding for mannitol L phosphate dehydrogenase (Lee andSaier, 1982) and trehalose 6 phosphate synthase (Kaasen at al., J.Bacteriology, 174:889-898, 1992). Through the subsequent action ofnative phosphatases in the cell or by the introduction and coexpressionof a specific phosphatase, these introduced genes will result in theaccumulation of either mannitol or trehalose, respectively, both ofwhich have been well documented as protective compounds able to mitigatethe effects of stress. Mannitol accumulation in transgenic tobacco hasbeen verified and preliminary results indicate that plants expressinghigh levels of this metabolite are able to tolerate an applied osmoticstress (Tarczynski et al., Science, 259:508-510, 1993, Tarczynski et alProc. Natl. Acad. Sci. USA, 89:1-5, 1993).

Similarly, the efficacy of other metabolites in protecting either enzymefunction (e.g., alanopine or propionic acid) or membrane integrity(e.g., alanopine) has been documented (Loomis et al., J. Expt. Zoology,252:9-15, 1989), and therefore expression of genes encoding for thebiosynthesis of these compounds might confer drought resistance in amanner similar to or complimentary to mannitol. Other examples ofnaturally occurring metabolites that are osmotically active and/orprovide some direct protective effect during drought and/or desiccationinclude fructose, erythritol (Coxson et al., Biotropica, 24:121-133,1992), sorbitol, dulcitol (Karsten et al., Botanica Marina, 35:11-19,1992), glucosylglycerol (Reed et al., J. Gen. Microbiology, 130:1-4,1984; Erdmann et al., J. Gen. Microbiology, 138:363-368, 1992), sucrose,stachyose (Koster and Leopold, Plant Physiol., 88:829-832, 1988;Blackman et al., Plant Physiol., 100:225-230, 1992), raflinose (BernalLugo and Leopold, Plant Physiol., 98:1207-1210, 1992), proline (Rensburgat al., J. Plant Physiol., 141:188-194, 1993), glycine betaine, ononitoland pinitol (Vernon and Bohnert, The EMBO J., 11:2077-2085, 1992).Continued growth and increased reproductive fitness during times ofstress may be augmented by introduction and expression of genes such asthose controlling the osmotically active compounds discussed above andother such compounds. Genes which promote the synthesis of anosmotically active polyol compound include genes which encode theenzymes mannitol 1 phosphate dehydrogenase, trehalose 6 phosphatesynthase and myoinositol 0 methyltransferase.

It is contemplated that the expression of specific proteins also mayincrease drought tolerance in Sugarcane. Three classes of LateEmbryogenic Abundant (LEA) Proteins have been assigned based onstructural similarities (see Dure et al., Plant Molecular Biology,12:475-486, 1989). All three classes of LEAs have been demonstrated inmaturing (e.g. desiccating) seeds. Within these 3 types of LEA proteins,the Type II (dehydrin type) have generally been implicated in droughtand/or desiccation tolerance in vegetative plant parts (e.g. Mundy andChua, The EMBO J., 7:2279-2286, 1988; Piatkowski et al., Plant Physiol.,94:1682-1688, 1990; Yamaguchi Shinozaki et al., Plant Cell Physiol.,33:217-224, 1992). Expression of a Type III LEA (HVA 1) in tobacco wasfound to influence plant height, maturity and drought tolerance(Fitzpatrick, Gen. Engineering News, 22:7, 1993). In rice, expression ofthe HVA 1 gene influenced tolerance to water deficit and salinity (Xu etal., Plant Physiol., 110:249-257, 1996). Expression of structural genesfrom any of the three LEA groups may therefore confer drought tolerance.Other types of proteins induced during water stress include thiolproteases, aldolases or transmembrane transporters (Guerrero et al.,Plant Molecular Biology, 15:11-26, 1990), which may confer variousprotective and/or repair type functions during drought stress. It alsois contemplated that genes that effect lipid biosynthesis and hencemembrane composition might also be useful in conferring droughtresistance in Sugarcane.

Many of these genes for improving drought resistance have complementarymodes of action. Thus, it is envisaged that combinations of these genesmight have additive and/or synergistic effects in improving droughtresistance in Sugarcane. Many of these genes also improve freezingtolerance (or resistance); the physical stresses incurred duringfreezing and drought are similar in nature and may be mitigated insimilar fashion. Benefits may be conferred via constitutive expressionof these genes; alternatively, one means of expressing these novel genesmay be through the use of a turgor induced promoter (such as thepromoters for the turgor induced genes described in Guerrero et al.,Plant Molecular Biology, 15:11-26, 1990 and Shagan et al., PlantPhysiol., 101:1397-1398, 1993 which are incorporated herein byreference). Spatial and temporal expression patterns of these genes mayenable plants to better withstand stress.

It is proposed that expression of genes that are involved with specificmorphological traits that allow for increased water extractions fromdrying soil would be of benefit. For example, introduction andexpression of genes that alter root characteristics may enhance wateruptake. It also is contemplated that expression of genes that enhancereproductive fitness during times of stress would be of significantvalue. For example, expression of genes that improve the synchrony ofpollen shed and receptiveness of the female flower parts, e.g., silks,would be of benefit. In addition it is proposed that expression of genesthat minimize kernel abortion during times of stress would increase theamount of grain to be harvested and hence be of value.

Given the overall role of water in determining yield, it is contemplatedthat enabling Sugarcane to utilize water more efficiently, through theintroduction and expression of novel genes, will improve overallperformance even when soil water availability is not limiting. Byintroducing genes that improve the ability of Sugarcane plants tomaximize water usage across a full range of stresses relating to wateravailability, yield stability or consistency of yield performance may berealized.

Polypeptides that may improve stress tolerance in Sugarcane under avariety of stress conditions include polypeptides involved in generegulation, such as serine/threonine-protein kinases, MAP kinases, MAPkinase kinases, and MAP kinase kinase kinases; polypeptides that act asreceptors for signal transduction and regulation, such as receptorprotein kinases; intracellular signaling proteins, such as proteinphosphatases, GTP binding proteins, and phospholipid signaling proteins;polypeptides involved in arginine biosynthesis; polypeptides involved inATP metabolism, including for example ATPase, adenylate transporters,and polypeptides involved in ATP synthesis and transport, polypeptidesinvolved in glycine betaine, jasmonic acid, flavonoid or steroidbiosynthesis; and hemoglobin. Enhanced or reduced activity of suchpolypeptides in Sugarcane plants comprising a Sugarcane mini-chromosomeor recombinant chromosome will provide changes in the ability of theplants to respond to a variety of environmental stresses, such aschemical stress, drought stress and pest stress.

Other polypeptides that may improve Sugarcane tolerance to cold orfreezing temperatures include polypeptides involved in biosynthesis oftrehalose or raffinose, polypeptides encoded by cold induced genes,fatty acyl desaturases and other polypeptides involved in glycerolipidor membrane lipid biosynthesis, which find use in modification ofmembrane fatty acid composition, alternative oxidase, calcium-dependentprotein kinases, LEA proteins or uncoupling protein.

Other polypeptides that may improve Sugarcane tolerance to heat includepolypeptides involved in biosynthesis of trehalose, polypeptidesinvolved in glycerolipid biosynthesis or membrane lipid metabolism (foraltering membrane fatty acid composition), heat shock proteins ormitochondrial NDK.

Other polypeptides that may improve Sugarcane tolerance to extremeosmotic conditions include polypeptides involved in prolinebiosynthesis.

Other polypeptides that may improve Sugarcane tolerance to droughtconditions include aquaporins, polypeptides involved in biosynthesis oftrehalose or wax, LEA proteins or invertase.

(iv) Disease Resistance

It is proposed that increased resistance (or tolerance) to diseases maybe realized through introduction of genes into Sugarcane. It is possibleto produce resistance to diseases caused by viruses, viroids, bacteria,fungi and nematodes. It also is contemplated that control of mycotoxinproducing organisms may be realized through expression of introducedgenes. Resistance can be affected through suppression of endogenousfactors that encourage disease-causing interactions, expression ofexogenous factors that are toxic to or otherwise provide protection frompathogens, or expression of factors that enhance Sugarcane's own defenseresponses.

Resistance to viruses may be produced through expression of novel genesin Sugarcane. For example, it has been demonstrated that expression of aviral coat protein in a modified plant can impart resistance toinfection of the plant by that virus and perhaps other closely relatedviruses (Cuozzo et al., Bio/Technology, 6:549-553, 1988, Hemenway atal., The EMBO J., 7:1273-1280, 1988, Abel et al., Science, 232:738-743,1986). It is contemplated that expression of antisense genes targeted atessential viral functions may also impart resistance to viruses. Forexample, an antisense gene targeted at the gene responsible forreplication of viral nucleic acid may inhibit replication and lead toresistance to the virus. It is believed that interference with otherviral functions through the use of antisense genes also may increaseresistance to viruses. Further, it is proposed that it may be possibleto achieve resistance to viruses through other approaches, including,but not limited to the use of satellite viruses.

It is proposed that increased resistance to diseases caused by bacteriaand fungi Sugarcane may be realized through introduction of novel genes.It is contemplated that genes encoding so called “peptide antibiotics,”pathogenesis related (PR) proteins, toxin resistance, or proteinsaffecting host pathogen interactions such as morphologicalcharacteristics will be useful. Peptide antibiotics are polypeptidesequences which are inhibitory to growth of bacteria and othermicroorganisms. For example, the classes of peptides referred to ascecropins and magainins inhibit growth of many species of bacteria andfungi. It is proposed that expression of PR proteins in Sugarcane may beuseful in conferring resistance to bacterial disease. These genes areinduced following pathogen attack on a host plant and have been dividedinto at least five classes of proteins (Bol, Linthorst, and Cornelissen,1990). Included amongst the PR proteins are beta 1,3 glucanases,chitinases, and osmotin and other proteins that are believed to functionin plant resistance to disease organisms. Other genes have beenidentified that have antifungal properties, e.g., UDA (stinging nettlelectin), or hevein (Broakaert et al., 1989; Barkai Golan et al., 1978).It is known that certain plant diseases are caused by the production ofphytotoxins. It is proposed that resistance to these diseases would beachieved through expression of a novel gene that encodes an enzymecapable of degrading or otherwise inactivating the phytotoxin. It alsois contemplated that expression of novel genes that alter theinteractions between the Sugarcane host and pathogen may be useful inreducing the ability of the disease organism to invade the tissues ofthe host plant, e.g., an increase in the waxiness of the leaf cuticle orother morphological characteristics.

Polypeptides useful for imparting improved disease responses toSugarcane include polypeptides encoded by cercosporin induced genes,antifungal proteins and proteins encoded by R-genes or SAR genes.

Agronomically important diseases in Sugarcane include but are notlimited to: pineapple disease of Sugarcane, pokkah boeng disease ofSugarcane, Sugarcane eye spot disease, Sugarcane leaf scald disease,Sugarcane mosaic virus disease, Sugarcane ratoon stunting disease,Sugarcane red rot Disease, Sugarcane rust Disease, Sugarcane smutdisease, Metarhizium anisopliae, Ustilago scitaminea, Colletotrichumfalcatum, Fusarium moniliformae, Cephalosporium sacchari, Certocystisparadoxa, Cercospora, Helminthosporium and Leptosphaeria, Puccinia.graminicolum, Puccinia aphanidermatum and Puccinia catenulatum,Xanthomonas albilineans, Leifsonia xyli, Sugarcane mosaic virus (SCMV)(Potyvirdae), Sugarcane bacilliform virus (SCBV) (Pararetroviridae),Sugarcane yellow leaf syndrome (YLS), and Sugarcane yellow loaf virus(ScYLV).

(v) Plant Agronomic Characteristics

Temperature also influences where Sugarcane can be grown. Within theareas where it is possible to grow Sugarcane, there are varyinglimitations on the maximal time it is allowed to grow to maturity and beharvested. For example, a variety to be grown in a particular area isselected for its ability to mature within the required period of timewith maximum possible yield. It is considered that genes that influencematurity can be identified and introduced into Sugarcane lines to createnew varieties adapted to different growing locations or the same growinglocation, but having improved yield at harvest. Expression of genes thatare involved in regulation of plant development may be especiallyuseful.

It is contemplated that genes may be introduced into Sugarcane thatwould improve standability and other plant growth characteristics.Expression of novel genes in Sugarcane which confer stronger stalks,improved root systems, or prevent or reduce ear droppage or shatteringwould be of great value to the farmer. It is proposed that introductionand expression of genes that increase the total amount ofphotoassimilate available by, for example, increasing light distributionand/or interception would be advantageous. In addition, the expressionof genes that increase the efficiency of photosynthesis and/or the leafcanopy would further increase gains in productivity. It is contemplatedthat expression of a phylochrone gene in Sugarcane may be advantageous.Expression of such a gene may reduce apical dominance, confersemidwarfism on a plant, or increase shade tolerance (U.S. Pat. No.5,268,526). Such approaches would allow for increased Sugarcanepopulations in the field.

(vi) Nutrient Utilization

The ability to utilize available nutrients may be a limiting factor ingrowth of Sugarcane. It is proposed that it would be possible to alternutrient uptake, tolerate pH extremes, mobilization through the plant,storage pools, and availability for metabolic activities by theintroduction of novel genes. These modifications would allow a Sugarcaneplant to more efficiently utilize available nutrients. It iscontemplated that an increase in the activity of, for example, an enzymethat is normally present in the plant and involved in nutrientutilization would increase the availability of a nutrient or decreasethe availability of an antinutritive factor. An example of such anenzyme would be phytase. It is further contemplated that enhancednitrogen utilization by Sugarcane is desirable. Expression of aglutamate dehydrogenase gene in plants, e.g., E. coli gdhA genes, maylead to increased fixation of nitrogen in organic compounds.Furthermore, expression of gdhA in Sugarcane may lead to enhancedresistance to the herbicide glufosinate by incorporation of excessammonia into glutamate, thereby detoxifying the ammonia. It also iscontemplated that expression of a novel gene may make a nutrient sourceavailable that was previously not accessible, e.g., an enzyme thatreleases a component of nutrient value from a more complex molecule,perhaps a macromolecule.

Polypeptides useful for improving nitrogen flow, sensing, uptake,storage and/or transport include those involved in aspartate, glutamineor glutamate biosynthesis, polypeptides involved in aspartate, glutamineor glutamate transport, polypeptides associated with the TOR (Target ofRapamycin) pathway, nitrate transporters, nitrate reductases, aminotransferases, ammonium transporters, chlorate transporters orpolypeptides involved in tetrapyrrole biosynthesis.

Polypeptides useful for increasing the rate of photosynthesis includephytochrome, ribulose bisphosphate carboxylase-oxygenase, Rubiscoactivase, photosystem I and II proteins, electron carriers, ATPsynthase, NADH dehydrogenase or cytochrome oxidase.

Polypeptides useful for increasing phosphorus uptake, transport orutilization include phosphatases or phosphate transporters.

(vii) Male Sterility

Male sterility is useful in the production of hybrid varieties ofSugarcane. It is proposed that male sterility may be produced throughexpression of novel genes. For example, it has been shown thatexpression of genes that encode proteins, RNAs, or peptides thatinterfere with development of the male inflorescence and/or gametophyteresult in male sterility. Chimeric ribonuclease genes that express inthe anthers of transgenic tobacco and oilseed rape have beendemonstrated to lead to male sterility (Mariani at al., Nature,347:737-741, 1990).

A number of mutations were discovered in maize that confer cytoplasmicmale sterility. One mutation in particular, referred to as T cytoplasm,also correlates with sensitivity to Southern corn leaf blight. A DNAsequence, designated TURF 13 (Levings, Science, 250:942-947, 1990), wasidentified that correlates with T cytoplasm. It is proposed that itwould be possible through the introduction of TURF 13 viatransformation, to separate male sterility from disease sensitivity. Asit is necessary to be able to restore male fertility for breedingpurposes and for grain production, it is proposed that genes encodingrestoration of male fertility also may be introduced.

(viii) Altered Nutritional Content

Genes may be introduced into Sugarcane to improve or alter the nutrientquality or content. Introduction of genes that alter the nutrientcomposition may greatly enhance the feed, food or forage value. Limitingessential amino acids may include lysine, methionine, tryptophan,threonine, valine, arginine, and histidine. The levels of theseessential amino acids may be elevated by mechanisms which include, butare not limited to, the introduction of genes to increase thebiosynthesis of the amino acids, decrease the degradation of the aminoacids, increase the storage of the amino acids in proteins, or increasetransport of the amino acids to particular tissues.

Polypeptides useful for providing increased protein quantity and/orquality include polypeptides involved in the metabolism of amino acidsin Sugarcane, particularly polypeptides involved in biosynthesis ofmethionine/cysteine and lysine, amino acid transporters, amino acidefflux carriers, seed storage proteins, proteases, or polypeptidesinvolved in phytic acid metabolism.

The protein composition of Sugarcane may be altered to improve thebalance of amino acids in a variety of ways including elevatingexpression of native proteins, decreasing expression of those with poorcomposition, changing the composition of native proteins, or introducinggenes encoding entirely new proteins possessing superior composition.

The introduction of genes that alter the oil content of Sugarcane mayalso be of value. Increases in oil content may result in increases inmetabolizable-energy-content. The introduced genes may encode enzymesthat remove or reduce rate-limitations or regulated steps in fatty acidor lipid biosynthesis. Such genes may include, but are not limited to,those that encode acetyl-CoA carboxylase, ACP-acyltransferase,alpha-ketoacyl-ACP synthase, or other well known fatty acid biosyntheticactivities. Other possibilities are genes that encode proteins that donot possess enzymatic activity such as acyl carrier protein. Genes maybe introduced that alter the balance of fatty acids present in the oilproviding a more healthful or nutritive feedstuff. The introduced DNAalso may encode sequences that block expression of enzymes involved infatty acid biosynthesis, altering the proportions of fatty acids presentin Sugarcane.

Genes may be introduced that enhance the nutritive value of Sugarcane,or of foods derived from Sugarcane by increasing the level of naturallyoccurring phytosterols, or by encoding for proteins to enable thesynthesis of phytosterols in Sugarcane. The phytosterols from theseSugarcane can be processed directly into foods, or extracted and used tomanufacture food products.

Genes may be introduced that enhance the nutritive value or energy valueof the starch component of Sugarcane, for example by altering the degreeof branching of starch molecules, resulting in improved utilization ofthe starch in biofuel or feedstock applications. Additionally, othermajor constituents of Sugarcane may be altered, including genes thataffect a variety of other nutritive, processing, or other qualityaspects. For example, pigmentation may be increased or decreased.

Carbohydrate metabolism may be altered, for example by increased sucroseproduction and/or transport. Polypeptides useful for affectingcarbohydrate metabolism include polypeptides involved in sucrose orstarch metabolism, carbon assimilation or carbohydrate transport,including, for example sucrose transporters or glucose/hexosetransporters, enzymes involved in glycolysis/gluconeogenesis, thepentose phosphate cycle, or raffinose biosynthesis, or polypeptidesinvolved in glucose signaling, such as SNF1 complex proteins.

Sugarcane may also possess sub-optimal quantities of vitamins,antioxidants or other nutraceuticals, requiring supplementation toprovide adequate nutritive value and ideal health value. Introduction ofgenes that enhance vitamin biosynthesis may be envisioned including, forexample, vitamins A, E, B12, choline, or the like. Mineral content mayalso be sub-optimal. Thus genes that affect the accumulation oravailability of compounds containing phosphorus, sulfur, calcium,manganese, zinc, or iron among others would be valuable.

Numerous other examples of improvements of Sugarcane may be used withthe invention. Introduction of DNA to accomplish this might includesequences that alter lignin production such as those that result in the“brown midrib” phenotype associated with superior feed value for cattle.Other genes may encode for enzymes that alter the structure ofextracellular carbohydrates, or that facilitate the degradation of thecarbohydrates so that it can be efficiently fermented into ethanol orother useful carbohydrates.

It may be desirable to modify the nutritional content of Sugarcane byreducing undesirable components such as fats, starches, etc. This may bedone, for example, by the use of exogenous nucleic acids that encodeenzymes which increase plant use or metabolism of such components sothat they are present at lower quantities. Alternatively, it may be doneby use of exogenous nucleic acids that reduce expression levels oractivity of native Sugarcane enzymes that synthesize such components.

Likewise the elimination of certain undesirable traits may improve thefood or feed value of Sugarcane. Many undesirable traits must currentlybe eliminated by special post-harvest processing steps and the degree towhich these can be engineered into Sugarcane prior to harvest andprocessing would provide significant value. Examples of such traits arethe elimination of anti-nutritionals such as phytates and phenoliccompounds which are commonly found in many crop species. Also, thereduction of fats, carbohydrates and certain phytohormones may bevaluable for the food and feed industries as they may allow a moreefficient mechanism to meet specific dietary requirements.

In addition to direct improvements in feed or food value, genes also maybe introduced which improve the processing of Sugarcane and improve thevalue of the products resulting from the processing. Novel genes thatincrease the efficiency and reduce the cost of such processing, forexample by decreasing the time required at a particular step, may alsofind use. Improving the value of products derived from processedSugarcane may include altering the quantity or quality of sugar, starch,oil, fiber, gluten, or other components. Elevation of sugar or starchmay be achieved through the identification and elimination of ratelimiting steps in starch and sugar biosynthesis by expressing increasedamounts of enzymes involved in biosynthesis or by decreasing levels ofthe other components resulting in proportional increases in sugar orstarch. In addition, Sugarcane can be modified by introducing orexpressing a gene or genes that produce novel products, such assecondary plant metabolites or pharmaceutical products, that could bepurified during the processing step. Using Sugarcane mini-chromosomes orrecombinant chromosomes to both introduce genes for new products andoptionally for improving processing steps could provide a cost effectiveoption to produce these novel products.

Oil is another product of processing, the value of which may be improvedby introduction and expression of genes. Oil properties may be alteredto improve its performance in the production and use of cooking oil,shortenings, lubricants or other oil-derived products or improvement ofits health attributes when used in the food-related applications. Novelfatty acids also may be synthesized which upon extraction can serve asstarting materials for chemical syntheses. The changes in oil propertiesmay be achieved by altering the type, level, or lipid arrangement of thefatty acids present in the oil. This in turn may be accomplished by theaddition of genes that encode enzymes that catalyze the synthesis ofnovel fatty acids (e.g. fatty acid elongases, desaturases) and thelipids possessing them or by increasing levels of native fatty acidswhile possibly reducing levels of precursors or breakdown products.Alternatively, DNA sequences may be introduced which slow or block stepsin fatty acid biosynthesis resulting in the increase in precursor fattyacid intermediates. Genes that might be added include desaturases,epoxidases, hydratases, dehydratases, or other enzymes that catalyzereactions involving fatty acid intermediates. Representative examples ofcatalytic steps that might be blocked include the desaturations fromstearic to oleic acid or oleic to linolenic acid resulting in therespective accumulations of stearic and oleic acids. Another example isthe blockage of elongation steps resulting in the accumulation of C8 toC12 saturated fatty acids.

Polypeptides useful for providing increased oil quantity and/or qualityinclude polypeptides involved in fatty acid and glycerolipidbiosynthesis, beta-oxidation enzymes, enzymes involved in biosynthesisof nutritional compounds, such as carotenoids and tocopherols.

Polypeptides involved in production of galactomannans orarabinogalactans are of interest for providing plants having increasedand/or modified reserve polysaccharides for use in food, pharmaceutical,cosmetic, paper and paint industries.

Polypeptides involved in modification of flavonoid/isoflavonoidmetabolism in plants include cinnamate-4-hydroxylase, chalcone synthaseor flavones synthase. Enhanced or reduced activity of such polypeptidesin Sugarcane plants comprising a Sugarcane mini-chromosome will providechanges in the quantity and/or speed of flavonoid metabolism in plantsand may improve disease resistance by enhancing synthesis of protectivesecondary metabolites or improving signaling pathways governing diseaseresistance.

Polypeptides involved in lignin biosynthesis are of interest forincreasing Sugarcane's resistance to lodging and for increasing theusefulness of plant materials as biofuels.

(ix) Production or Assimilation of Chemicals or Biologicals

It may further be considered that Sugarcane plants comprising aSugarcane mini-chromosome or recombinant chromosome prepared inaccordance with the invention may be used for the production ormanufacturing of useful biological compounds that were either notproduced at all, or not produced at the same level, in the Sugarcaneplant previously. Alternatively, plants produced in accordance with theinvention may be made to metabolize or absorb and concentrate certaincompounds, such as hazardous wastes, thereby allowing bioremediation ofthese compounds.

The novel Sugarcane plants producing these compounds are made possibleby the introduction and expression of one or potentially many genes withthe constructs provided by the invention. The vast array ofpossibilities include but are not limited to any biological compoundwhich is presently produced by any organism such as proteins, nucleicacids, primary and intermediary metabolites, carbohydrate polymers,enzymes for uses in bioremediation, enzymes for modifying pathways thatproduce secondary plant metabolites such as falconoid or vitamins,enzymes that could produce pharmaceuticals, and for introducing enzymesthat could produce compounds of interest to the manufacturing industrysuch as specialty chemicals and plastics. The compounds may be producedby the Sugarcane plant, extracted upon harvest and/or processing, andused for any presently recognized useful purpose such aspharmaceuticals, fragrances, and industrial enzymes to name a few.

(x) Other Characteristics

Cell cycle modification: Polypeptides encoding cell cycle enzymes andregulators of the cell cycle pathway are useful for manipulating growthrate in Sugarcane to provide early vigor and accelerated maturation.Improvements in quality traits, such as seed oil content, may also beobtained by expression of cell cycle enzymes and cell cycle regulators.Polypeptides of interest for modification of the cell cycle pathwayinclude cycling and EIF5α pathway proteins, polypeptides involved inpolyamine metabolism, polypeptides which act as regulators of the cellcycle pathway, including cyclin-dependent kinases (CDKs), CDK-activatingkinases, cell cycle-dependent phosphatases, CDK-inhibitors, Rb andRb-binding proteins, or transcription factors that activate genesinvolved in cell proliferation and division, such as the E2F family oftranscription factors, proteins involved in degradation of cyclins, suchas cullins, and plant homologs of tumor suppressor polypeptides.

Plant growth regulators: Polypeptides involved in production ofsubstances that regulate the growth of various plant tissues are ofinterest in the present invention and may be used to provide Sugarcaneplants comprising a Sugarcane mini-chromosome having alteredmorphologies and improved plant growth and development profiles leadingto improvements in yield and stress response. Of particular interest arepolypeptides involved in the biosynthesis, or degradation of plantgrowth hormones, such as gibberellins, brassinosteroids, cytokinins,auxins, ethylene or abscisic acid, and other proteins involved in theactivity, uptake and/or transport of such polypeptides, including forexample, cytokinin oxidase, cytokinin/purine permeases, F-box proteins,G-proteins or phytosulfokines.

Transcription factors in plants: Transcription factors play a key rolein plant growth and development by controlling the expression of one ormore genes in temporal, spatial and physiological specific patterns.Enhanced or reduced activity of such polypeptides in Sugarcane plantscomprising a Sugarcane mini-chromosome will provide significant changesin gene transcription patterns and provide a variety of beneficialeffects in plant growth, development and response to environmentalconditions. Transcription factors of interest include, but are notlimited to myb transcription factors, including helix-turn-helixproteins, homeodomain transcription factors, leucine zippertranscription factors, MADS transcription factors, transcription factorshaving AP2 domains, zinc finger transcription factors, CCAAT bindingtranscription factors, ethylene responsive transcription factors,transcription initiation factors or UV damaged DNA binding proteins.

Homologous recombination: Increasing the rate of homologousrecombination in Sugarcane is useful for accelerating the introgressionof transgenes into breeding varieties by backcrossing, and to enhancethe conventional breeding process by allowing rare recombinants betweenclosely linked genes in phase repulsion to be identified more easily.Polypeptides useful for expression in plants to provide increasedhomologous recombination include polypeptides involved in mitosis and/ormeiosis, DNA replication, nucleic acid metabolism, DNA repair pathwaysor homologous recombination pathways including for example,recombinases, nucleases, proteins binding to DNA double-strand breaks,single-strand DNA binding proteins, strand-exchange proteins,resolvases, ligases, helicases and polypeptide members of the RAD52epistasis group.

Enhanced Biofuel Conversion

Biofuels may be produced from the conversion of Sugarcane biomass intoliquid or gaseous fuels by converting the biomass into sugars, or bydirect extraction of sugars, that can be fermented or chemicallyconverted to form a biofuel. Biofuels can also be generated byextracting oils from the biomass. Exemplary biofuels are ethanol,propanol, butanol, methanol, methane, 2,5-dimethylfurqan, dimethylether, biodiesel (short chain acid alkyl esters), biogasoline, paraffins(alkanes), other hydrocarbons or co-products of hydrogen.

The invention provides for Sugarcane mini-chromosomes or recombinantchromosomes expressing at least one gene that enhances or increasessugar production or extractability, enhances or increases biomass,enhances the conversion of biomass to sugars or enhances sugarfermentation to biofuels. It may further be considered that a modifiedSugarcane plant prepared in accordance with the invention may be used asbiomass for the production of biofuels or the plant may facilitateconversion of biomass to sugars or facilitate fermentation of sugars tobiofuels.

Enzymes that may be useful for biofuel production include those thatbreak down glucans. In some embodiments, the enzymes are selected fromthe group consisting of: endo-β(1,4)-glucanase, cellobiohydrolase,β-glucosidase, α/β-glucosidase, mixed-linked glucanase,endo-β(1,3)-glucanase, exo-β(1,3)-glucanase and β-(1,6)-glucanase. Inother embodiments the enzymes break down xyloglucans, xylans, mannans orlignins.

The enzyme genes may be controlled by inducible promoters that may beinactive until a desired time, such as at harvest or when the plant isadded to the biofuels process (e.g. inactive at physiologicalconditions, then activated by heat or pH), or sequestered by subcellularlocalization. The enzymes may also be controlled by a tissue-specificpromoter which may be active only in specific tissues (e.g. seeds orleaves).

Non-Protein-Expressing Exogenous Nucleic Acids

Sugarcane plants with decreased expression of a gene of interest canalso be achieved, for example, by expression of antisense nucleic acids,dsRNA or RNAi, catalytic RNA such as ribozymes, sense expressionconstructs that exhibit cosuppression effects, aptamers or zinc fingerproteins.

Antisense RNA reduces production of the polypeptide product of thetarget messenger RNA, for example by blocking translation throughformation of RNA:RNA duplexes or by inducing degradation of the targetmRNA. Antisense approaches are a way of preventing or reducing genefunction by targeting the genetic material as disclosed in U.S. Pat.Nos. 4,801,540; 5,107,065; 5,759,829; 5,910,444; 6,184,439; and6,198,026, all of which are incorporated herein by reference. In oneapproach, an antisense gene sequence is introduced that is transcribedinto antisense RNA that is complementary to the target mRNA. Forexample, part or all of the normal gene sequences are placed under apromoter in inverted orientation so that the complementary strand istranscribed into a non-protein expressing antisense RNA. The promoterused for the antisense gene may influence the level, timing, tissue,specificity, or inducibility of the antisense inhibition.

Autonomous Sugarcane mini-chromosomes or recombinant chromosomes maycomprise exogenous DNA flanked by recombination sites, for example lox-Psites, that can be recognized by a recombinase, e.g. Cre, and removedfrom the Sugarcane mini-chromosome or recombinant chromosome. In caseswhere there is a homologous recombination site or sites in the hostgenomic DNA, the exogenous DNA excised from the Sugarcanemini-chromosome or recombinant chromosome may be integrated into thegenome at one of the specific recombination sites and the DNA flanked bythe recombination sites will become integrated into the host DNA. Theuse of a Sugarcane mini-chromosome or recombinant chromosome as aplatform for DNA excision or for launching such DNA integration into thehost genome may include in vivo induction of the expression of arecombinase encoded in the genomic DNA of a transgenic host, or in aSugarcane mini-chromosome or recombinant chromosome.

RNAi gene suppression in plants by transcription of a dsRNA is describedin U.S. Pat. No. 6,506,559, U.S. patent application Publication No.2002/0168707, WO 98/53083, WO 99/53050 and WO 99/61631, all of which areincorporated herein by reference. The double-stranded RNA or RNAiconstructs can trigger the sequence-specific degradation of the targetmessenger RNA. Suppression of a gene by RNAi can be achieved using arecombinant DNA construct having a promoter operably linked to a DNAelement comprising a sense and anti-sense element of a segment ofgenomic DNA of the gene, e.g., a segment of at least about 23nucleotides, optionally about 50 to 200 nucleotides where the sense andanti-sense DNA components can be directly linked or joined by an intronor artificial DNA segment that can form a loop when the transcribed RNAhybridizes to form a hairpin structure.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of the target gene or genes or facilitate molecularreactions. Ribozymes are targeted to a given sequence by hybridizationof sequences within the ribozyme to the target mRNA. Two stretches ofhomology are required for this targeting, and these stretches ofhomologous sequences flank the catalytic ribozyme structure. It ispossible to design ribozymes that specifically pair with virtually anytarget mRNA and cleave the target mRNA at a specific location, therebyinactivating it. A number of classes of ribozymes have been identified.One class of ribozymes is derived from a number of small circular RNAsthat are capable of self-cleavage and replication in plants. The RNAsreplicate either alone (viroid RNAs) or with a helper virus (satelliteRNAs). Examples include Tobacco Ringspot Virus (Prody et al. Science,231:1577-1580, 1986), Avocado Sunblotch Viroid (Palukaitis et al.,Virology, 99:145-151, 1979; Symons, Nucl. Acids Res., 9:6527-6537,1981), and Lucerne Transient Streak Virus (Forster and Symons, Cell,49:211-220, 1987), and the satellite RNAs from velvet tobacco mottlevirus, Solanum nodiflorum mottle virus and subterranean clover mottlevirus. The design and use of target RNA-specific ribozymes is describedin Haseloff, et al., Nature 334:585-591 (1988). Several differentribozyme motifs have been described with RNA cleavage activity (Symons,Annu. Rev. Biochem., 61:641-671, 1992). Other suitable ribozymes includesequences from RNase P with RNA cleavage activity (Yuan et al., Proc.Natl. Acad. Set. USA, 89:8006-8010, 1992; Yuan and Allman, Science,263:1269-1273, 1994; U.S. Pat. Nos. 5,168,053 and 5,624,824), hairpinribozyme structures (Berzal-Herranz et al., Genes and Devel., 6:129-134,1992; Chowrira et al., J. Biol. Chem., 269:25856-25864, 1994) andHepatitis Delta virus based ribozymes (U.S. Pat. No. 5,625,047). Thegeneral design and optimization of ribozyme directed RNA cleavageactivity has been discussed in detail (Haseloff and Gerlach, 1988,Nature. 1988 Aug. 18; 334(6183):585-91, Chowrira et al., J. Biol. Chem.,269:25856-25864, 1994).

Another method of reducing protein expression utilizes the phenomenon ofcosuppression or gene silencing (for example, U.S. Pat. Nos. 6,063,947;5,686,649; or 5,283,184; cach of which is incorporated herein byreference). Cosuppression of an endogenous gene using a full-length cDNAsequence as well as a partial cDNA sequence are known (for example,Napoli et al., Plant Cell 2:279-289 [1990]; van der Krol et al., PlantCell 2:291-299 [1990]; Smith et al., Mol. Gen. Genetics 224:477-481[1990]). The phenomenon of cosuppression has also been used to inhibitplant target genes in a tissue-specific manner.

In some embodiments, nucleic acids from one species of plant areexpressed in another species of plant to effect cosuppression of ahomologous gene. The introduced sequence generally will be substantiallyidentical to the endogenous sequence intended to be repressed, forexample, about 65%, 80%, 85%, 90%, 95% or even 98% or greater identical.Higher identity may result in a more effective repression of expressionof the endogenous sequence. A higher identity in a shorter than fulllength sequence compensates for a longer, less identical sequence.Furthermore, the introduced sequence need not have the same intron orexon pattern, and identity of non-coding segments will be equallyeffective. Generally, where inhibition of expression is desired, sometranscription of the introduced sequence occurs. The effect may occurwhere the introduced sequence contains no coding sequence per se, butonly intron or untranslated sequences homologous to sequences present inthe primary transcript of the endogenous sequence.

Yet another method of reducing protein activity is by expressing nucleicacid ligands, so-called aptamers, which specifically bind to theprotein. Aptamers may be obtained by the SELEX (Systematic Evolution ofLigands by Exponential Enrichment) method. See U.S. Pat. No. 5,270,163,incorporated herein by reference. In the SELEX method, a candidatemixture of single stranded nucleic acids having regions of randomizedsequence is contacted with the protein and those nucleic acids having anincreased affinity to the target are selected and amplified. Afterseveral iterations a nucleic acid with optimal affinity to thepolypeptide is obtained and is used for expression in modified plants.

A zinc finger protein that binds a polypeptide-encoding sequence or itsregulatory region is also used to alter expression of the nucleotidesequence. Transcription of the nucleotide sequence may be reduced orincreased. Zinc finger proteins are, for example, described in Beerli etal. (1998) PNAS 95:14628-14633., or in WO 95/19431, WO 98/54311, or WO96/06166, all incorporated herein by reference.

Other examples of non-protein expressing sequences specificallyenvisioned for use with the invention include tRNA sequences, forexample, to alter codon usage, and rRNA variants, for example, which mayconfer resistance to various agents such as antibiotics.

It is contemplated that unexpressed DNA sequences, including novelsynthetic sequences, could be introduced into Sugarcane cells asproprietary “labels” of those cells and plants and seeds thereof. Itwould not be necessary for a label DNA element to disrupt the functionof a gene endogenous to the host organism, as the sole function of thisDNA would be to identify the origin of the organism. For example, onecould introduce a unique DNA sequence into a Sugarcane plant and thisDNA element would identify all cells, plants, and progeny of these cellsas having arisen from that labeled source. It is proposed that inclusionof label DNAs would enable one to distinguish proprietary germplasm orgermplasm derived from such, from unlabelled germplasm.

Exemplary Plant Promoters, Regulatory Sequences and Targeting Sequences

Exemplary classes of plant promoters are described below.

Constitutive Expression promoters: Exemplary constitutive expressionpromoters include the ubiquitin promoter (e.g., sunflower-Binet et al.Plant Science 79: 87-94 (1991); maize—Christensen et al. Plant Molec.Biol. 12: 619-632 (1989); and Arabidopsis—Callis et al., J. Biol. Chem.265: 12486-12493 (1990) and Norris et al., Plant Mol. Biol. 21: 895-906(1993)); the CaMV 35S promoter (U.S. Pat. Nos. 5,858,742 and 5,322,938);or the actin promoter (e.g., rice—U.S. Pat. No. 5,641,876; McElroy etal. Plant Cell 2: 163-171 (1990), McElroy et al. Mol. Gen. Genet. 231:150-160 (1991), and Chibbar et al. Plant Cell Rep. 12: 506-509 (1993)).Exemplary promoters for use in Sugarcane include the maize polyubiquitin1 (Mubi-1) and the Sugarcane polyubiquitin 9 (SCubi9) promoters (Wang ML, Goldstein C, Su W, Moore P H, Albert H H. Production of biologicallyactive GM-CSF in Sugarcane: a secure biofactory. Transgenic Res. 2005,14:167-78); and the Sugarcane polyubiquitin 4 (ubi4) promoter (Wei H,Wang M L, Moore P H, Albert H H. Comparative expression analysis of twoSugarcane polyubiquitin promoters and flanking sequences in transgenicplants. J Plant Physiol. 2003, 160:1241-51).

Inducible Expression promoters: Exemplary inducible expression promotersinclude the chemically regulatable tobacco PR-1 promoter (e.g.,tobacco—U.S. Pat. No. 5,614,395; Arabidopsis-Lebel et al., Plant J. 16:223-233 (1998); maize—U.S. Pat. No. 6,429,362). Various chemicalregulators may be employed to induce expression, including thebenzothiadiazole, isonicotinic acid, and salicylic acid compoundsdisclosed in U.S. Pat. Nos. 5,523,311 and 5,614,395. Other promotersinducible by certain alcohols or ketones, such as ethanol, include, forexample, the alcA gene promoter from Aspergillus nidulans (Caddick etal. (1998) Nat. Biotechnol 16:177-180). A glucocorticoid-mediatedinduction system is described in Aoyama and Chua (1997) The PlantJournal 11: 605-612 wherein gene expression is induced by application ofa glucocorticoid, for example a dexamethasone. Another class of usefulpromoters are water-deficit-inducible promoters, e.g. promoters whichare derived from the 5′ regulatory region of genes identified as a heatshock protein 17.5 gene (HSP 17.5), an HVA22 gene (HVA22), and acinnamic acid 4-hydroxylase (CA4H) gene of Zea mays. Anotherwater-deficit-inducible promoter is derived from the rab-17 promoter asdisclosed by Vilardell et al., Plant Molecular Biology, 17(5):985-993,1990. See also U.S. Pat. No. 6,084,089 which discloses cold induciblepromoters, U.S. Pat. No. 6,294,714 which discloses light induciblepromoters, U.S. Pat. No. 6,140,078 which discloses salt induciblepromoters, U.S. Pat. No. 6,252,138 which discloses pathogen induciblepromoters, and U.S. Pat. No. 6,175,060 which discloses phosphorusdeficiency inducible promoters.

As another example, numerous wound-inducible promoters have beendescribed (e.g. Xu et al. Plant Molec. Biol. 22: 573-588 (1993),Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, PlantMolec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22:129-142 (1993), Warner et al. Plant J. 3: 191-201 (1993)). Logemann etal., describe 5′ upstream sequences of the potato wunl gene. Xu et al.show that a wound-inducible promoter from the dicotyledon potato (pin2)is active in the monocotyledon rice. Rohrmeier & Lehle describe maizeWipl cDNA which is wound induced and which can be used to isolate thecognate promoter. Firek et al. and Warner et al. have described awound-induced gene from the monocotyledon Asparagus officinalis, whichis expressed at local wound and pathogen invasion sites.

Tissue-Specific Promoters: Exemplary promoters that express genes onlyin certain Sugarcane tissues are useful according to the presentinvention. For example root specific expression may be attained usingthe promoter of the maize metallothionein-like (MTL) gene described byde Framond (FEBS 290: 103-106 (1991)) and also in U.S. Pat. No.5,466,785, incorporated herein by reference. U.S. Pat. No. 5,837,848discloses a root specific promoter. Another exemplary promoter conferspith-preferred expression (see Int'l. Pub. No. WO 93/07278, hereinincorporated by reference, which describes the maize trpA gene andpromoter that is preferentially expressed in pith cells). Leaf-specificexpression may be attained, for example, by using the promoter for amaize gene encoding phosphoenol carboxylase (PEPC) (sce Hudspeth &Grula, Plant Molee Biol 12: 579-589 (1989)). Pollen-specific expressionmay be conferred by the promoter for the maize calcium-dependent proteinkinase (CDPK) gene which is expressed in pollen cells (WO 93/07278).U.S. Pat. Appl. Pub. No. 20040016025 describes tissue-specificpromoters. Pollen-specific expression may be conferred by the tomatoLAT52 pollen-specific promoter (Bate et. al., Plant Mol Riol. 1998 July;37(5):859-69).

See also U.S. Pat. No. 6,437,217 which discloses a root-specific maizeRS81 promoter, U.S. Pat. No. 6,426,446 which discloses a root specificmaize RS324 promoter, U.S. Pat. No. 6,232,526 which discloses aconstitutive maize A3 promoter, U.S. Pat. No. 6,177,611 which disclosesconstitutive maize promoters, U.S. Pat. No. 6,433,252 which discloses amaize L3 oleosin promoter that are aleurone and seed coat-specificpromoters, U.S. Pat. No. 6,429,357 which discloses a constitutive riceactin 2 promoter and intron, U.S. patent application Pub. No.20040216189 which discloses an inducible constitutive leaf specificmaize chloroplast aldolase promoter.

Optionally a plant transcriptional terminator can be used in place ofthe plant-expressed gene native transcriptional terminator. Exemplarytranscriptional terminators are those that are known to function inplants and include the CaMV 35S terminator, the tm1 terminator, thenopaline synthase terminator and the pea rbcS E9 terminator. These canbe used in both monocotyledons and dicotyledons.

Various intron sequences have been shown to enhance expression,particularly in monocotyledonous cells. For example, the introns of themaize Adh1 gene have been found to significantly enhance expression.Intron 1 was found to be particularly effective and enhanced expressionin fusion constructs with the chloramphenicol acetyltransferase gene(Callis et al., Genes Develop. 1: 1183-1200 (1987)). The intron from themaize bronze1 gene also enhances expression. Intron sequences have beenroutinely incorporated into plant transformation vectors, typicallywithin the non-translated leader. U.S. Patent Application Publication2002/0192813 discloses 5′, 3′ and intron elements useful in the designof effective plant expression vectors.

A number of non-translated leader sequences derived from viruses arealso known to enhance expression, and these are particularly effectivein dicotyledonous cells. Specifically, leader sequences from TobaccoMosaic Virus (TMV, the “omega-sequence”), Maize Chlorotic Mottle Virus(MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effectivein enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15:8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)).Other leader sequences known in the art include, but are not limited to:picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′noncoding region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. PNAS USA86:6126-6130 (1989)); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al., 1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20); human immunoglobulin heavy-chainbinding protein (BiP) leader, (Macejak, D. G., and Sarnow, P., Nature353: 90-94 (1991); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L.,Nature 325:622-625 (1987); tobacco mosaic virus leader (TMV), (Gallie etal., Molecular Biology of RNA, pages 237-256 (1989); or Maize ChloroticMottle Virus leader (MCMV) (Lommel et al., Virology 81:382-385 (1991).See also, Della-Cioppa et al., Plant Physiology 84:965-968 (1987).

A minimal promoter may also be incorporated. Such a promoter has lowbackground activity in plants when there is no transactivator present orwhen enhancer or response element binding sites are absent. Oneexemplary minimal promoter is the Bzl minimal promoter, which isobtained from the bronze1 gene of maize. Roth et al., Plant Cell 3: 317(1991). A minimal promoter may also be created by use of a syntheticTATA element. The TATA element allows recognition of the promoter by RNApolymerase factors and confers a basal level of gene expression in theabsence of activation (see generally, Mukumoto (1993) Plant Mol Biol 23:995-1003; Green (2000) Trends Biochem Sci 25: 59-63).

Sequences controlling the targeting of gene products also may beincluded. For example, the targeting of gene products to the chloroplastis controlled by a signal sequence found at the amino terminal end ofvarious proteins which is cleaved during chloroplast import to yield themature protein (e.g. Comai et al. J. Biol. Chem. 263: 15104-15109(1988)). These signal sequences can be fused to heterologous geneproducts to effect the import of heterologous products into thechloroplast (van den Broeck, et al. Nature 313: 358-363 (1985)). DNAencoding for appropriate signal sequences can be isolated from the 5′end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSPsynthase enzyme, the GS2 protein or many other proteins which are knownto be chloroplast localized. Other gene products are localized to otherorganelles such as the mitochondrion and the peroxisome (e.g. Unger etal. Plant Molec. Biol. 13: 411-418 (1989)). Examples of sequences thattarget to such organelles are the nuclear-encoded ATPases or specificaspartate amino transferase isoforms for mitochondria. Targetingcellular protein bodies has been described by Rogers et al. (Proc. Natl.Acad. Sci. USA 82: 6512-6516 (1985)). In addition, amino terminal andcarboxy-terminal sequences are responsible for targeting to the ER, theapoplast, and extracellular secretion from aleurone cells (Koehler & Ho,Plant Cell 2: 769-783 (1990)). Additionally, amino terminal sequences inconjunction with carboxy terminal sequences are responsible for vacuolartargeting of gene products (Shinshi et al. Plant Molec. Biol. 14:357-368 (1990)).

Another possible element which may be introduced is a matrix attachmentregion element (MAR), such as the chicken lysozyme A element, which canbe positioned around an expressible gene of interest to effect anincrease in overall expression of the gene and diminish positiondependent effects upon incorporation into the plant genome (Stiefet al.,Nature, 341:343, 1989; Phi-Van et al., Mol. Cell. Biol.,10:2302-2307.1990).

Use of Non-Plant Promoter Regions Isolated from Drosophila melanogasterand Saccharomyces cerevisiae to Express Genes in Plants

The promoter in the Sugarcane mini-chromosome or recombinant chromosomeof the present invention can be derived from plant or non-plant species.In one embodiment, the nucleotide sequence of the promoter is derivedfrom non-plant species for the expression of genes in plant cells. Inone embodiment, the non-plant promoters are constitutive or induciblepromoters derived from insect, e.g., Drosophila melanogaster or yeast,e.g., Saccharomyces cerevisiae. Table 2 lists nonlimiting examples ofpromoters from Drosophila melanogaster and Saccharomyces cerevisiae thatcan be used to derive the examples of non-plant promoters in the presentinvention. Promoters derived from any animal, protist, or fungi are alsocontemplated. SEQ ID NOS: 222-241, or fragments, mutants, hybrid ortandem promoters thereof, are examples of promoter sequences derivedfrom Drosophila melanogaster or Saccharomyces cerevisiae. Thesenon-plant promoters can be operably linked to nucleic acid sequencesencoding polypeptides or non-protein-expressing sequences including, butnot limited to, antisense RNA and ribozymes, to form nucleic acidconstructs, vectors, and host cells (prokaryotic or eukaryotic),comprising the promoters.

TABLE 2 Drosophila melanogaster Promoters (Information obtained from theFlybase Web Site at http://flybase.bio.indiana.edu/ which is a databaseof the Drosophila Genome) SEQ Standard promoter Chromo- ID NO: SSymFlybase ID gen

 name Gene Product some 222 Pgd FBgn0004654 Phosphogluconate6-phosphogluconate X dehydrogenase dehydrogenase 223 Grim FBgn0015946grim grim-P138 3 224 Uro FBgn0003961 Urate oxidase Uro-P1 2 225 SnaFBgn0003448 Snail sna-P1 2 226 Rh3 FBgn0003249 Rhodopsin 3 Rh3 3 227Lsp-1 γ FBgn0002564 Larval serum protein 1 Lsp1γ-P1 3 Saccharomycescerevisiae Promoters (Information obtained from the Saccharomyces GenomeDatabase Web site at http://www.yeastgenome.org/SearchContents.shtml SeqSystematic Standard promoter Chromo- No. SSymbol Name gen

 name Gene Product some 228 Tef-2 YBR118W TEF2 (Translation Translationelongation 2 elongation factor fact EF-1 alpha promoter) 229 Leu-1YGL009C LEU1 (LEUcine isopropylmalate 7 biosynthesis) isomerase 230Met16 YPR167C METhionine 3′phosphoadenylyl- 16 requiring sulfatereductase 231 Leu-2 YCL018W LEU2 (leucine beta-IPM (isopropylmalate 3biosynthesis) dehydrogenase) 232 His-4 YCL030C HIS4 (HIStidinehistidinol 3 requiring) dehydrogenase 233 Met-2 YNL277W MET2 (methionineL-homoserine-O- 14 requiring) acetyltransferase 234 Ste-3 YKL178C STE3(alias DAF2 a-factor receptor 11 Sterile) 235 Arg-1 YOL058W ARG1(aliasARG10 arginosuccinate 15 ARGinine requiring) synthetase 236 Pgk-1YCR012W PGK1 (phosphoglycerate phosphoglycerate kinase 3 kinase) 237GPD-1 YDL022W GPD1 (alias glycerol-3-phosphate 4 DAR1/HOR1/OSG1/O

dehydrogenase R5: glycerol-3-phospha

dehydrogenase activity 238 ADH1 YOL086C ADH1 (alias ADC1) alcoholdehydrogenase 15 239 GPD-2 YOL059W GPD2 (alias GPD3:glycerol-3-phosphate 15 glycerol-3-phosphate dehydrogenase dehydrogenaseactivity 240 Arg-4 YHR018C ARGinine requiring argininosuccinate lyase 8241 Yat-1 YAR035W YAT-1(carnitine carnitine 1 acetyltransferase)acetyltransferase

indicates data missing or illegible when filed

In the Sugarcane mini-chromosomes or recombinant chromosome of thepresent invention, the promoter may be a mutant of the promoters havinga substitution, deletion, and/or insertion of one or more nucleotides inthe nucleic acid sequence of SEQ ID NOS: 222-241, hybrid or tandempromoters.

The techniques used to isolate or clone a nucleic acid sequencecomprising a promoter of interest are known in the art and includeisolation from genomic DNA. The cloning procedures may involve excisionor amplification, for example by polymerase chain reaction, andisolation of a desired nucleic acid fragment comprising the nucleic acidsequence encoding the promoter, insertion of the fragment into a vectormolecule, and incorporation of the recombinant vector into the plantcell.

DEFINITIONS

The term “adchromosomal” Sugarcane plant or plant part as used hereinmeans a Sugarcane plant or plant part that contains functional, stableand autonomous Sugarcane mini-chromosomes. Adchromosomal Sugarcaneplants or plant parts may be chimeric or not chimeric (chimeric meaningthat Sugarcane mini-chromosomes are only in certain portions of theplant, and are not uniformly distributed throughout the plant). Anadchromosomal Sugarcane plant cell contains at least one functional,stable and autonomous Sugarcane mini-chromosome.

The term “autonomous” as used herein means that when delivered to plantcells, at least some Sugarcane mini-chromosomes are transmitted throughmitotic division to daughter cells and are episomal in the daughterplant cells, i.e. are not chromosomally integrated in the daughter plantcells. Daughter plant cells that contain autonomous mini-chromosomes canbe selected for further replication using, for example, selectable orscreenable markers. During the introduction into a cell of amini-chromosome, or during subsequent stages of the cell cycle, theremay be chromosomal integration of some portion or all of the DNA derivedfrom a mini-chromosome in some cells. The mini-chromosome is stillcharacterized as autonomous despite the occurrence of such events if aplant may be regenerated that contains episomal descendants of themini-chromosome, optionally distributed throughout its parts, or ifgametes or progeny can be derived from the plant that contain episomaldescendants of the mini-chromosome distributed through its parts.

As used herein, a “centromere” is any DNA sequence that confers anability to segregate to daughter cells through cell division. In onecontext, this sequence may produce a transmission efficiency to daughtercells ranging from about 1% to about 100%, including to about 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 95% of daughter cells.Variations in transmission efficiency may find important applicationswithin the scope of the invention; for example, mini-chromosomescarrying centromeres that confer 100% stability could be maintained inall daughter cells without selection, while those that confer 1%stability could be temporarily introduced into a transgenic organism,but be eliminated when desired. In particular embodiments of theinvention, the centromere may confer stable transmission to daughtercells of a nucleic acid sequence, including a recombinant constructcomprising the centromere, through mitotic or meiotic divisions,including through both meiotic and meiotic divisions. A plant centromereis not necessarily derived from plants, but has the ability to promoteDNA transmission to daughter plant cells.

As used herein, the term “circular permutations” refer to variants of asequence that begin at base n within the sequence, proceed to the end ofthe sequence, resume with base number one of the sequence, and proceedto base n−1. For this analysis, n may be any number less than or equalto the length of the sequence. For example, circular permutations of thesequence ABCD are: ABCD, BCDA, CDAB, and DABC.

The term “co-delivery” as used herein refers to the delivery of twonucleic acid segments to a cell. In co-delivery of plant growth inducinggenes and mini-chromosomes, the two nucleic acid segments are deliveredsimultaneously using the same delivery method. Alternatively, thenucleic acid segment containing the growth inducing gene, optionally aspart of an episomal vector, such as a viral vector or a plasmid vector,may be delivered to the plant cells before or after delivery of themini-chromosome, and the mini-chromosome may carry an exogenous nucleicacid that induces expression of the earlier-delivered growth inducinggene. In this embodiment, the two nucleic acid segments may be deliveredseparately at different times provided the encoded growth inducingfactors are functional during the appropriate time period.

The term “coding sequence” is defined herein as a nucleic acid sequencethat is transcribed into mRNA which is translated into a polypeptidewhen placed under the control of promoter sequences. The boundaries ofthe coding sequence are generally determined by the ATG start codonlocated at the start of the open reading frame, near the 5′ end of themRNA, and TAG, TGA or TAA stop codons at the end of the coding sequence,near the 3′ end of the mRNA, and in some cases, a transcriptionterminator sequence located just downstream of the open reading frame atthe 3′ end of the mRNA. A coding sequence can include, but is notlimited to, genomic DNA, cDNA, semisynthetic, synthetic, or recombinantnucleic acid sequences.

As used herein the term “consensus” refers to a nucleic acid sequencederived by comparing two or more related sequences. A consensus sequencedefines both the conserved and variable sites between the sequencesbeing compared. Any one of the sequences used to derive the consensus orany permutation defined by the consensus may be useful in theconstruction of mini-chromosomes.

The term “exogenous” when used in reference to a nucleic acid, forexample, is intended to refer to any nucleic acid that has beenintroduced into a recipient cell, regardless of whether the same orsimilar nucleic acid is already present in such a cell. Thus, as anexample, “exogenous DNA” can include an additional copy of DNA that isalready present in the plant cell, DNA from another plant, DNA from adifferent organism, or a DNA generated externally, such as a DNAsequence containing an antisense message of a gene, or a DNA sequenceencoding a synthetic or modified version of a gene. An “exogenous gene”can be a gene not normally found in the host genome in an identicalcontext, or an extra copy of a host gene. The gene may be isolated froma different species than that of the host genome, or alternatively,isolated from the host genome but operably linked to one or moreregulatory regions which differ from those found in the unaltered,native gene.

The term “functional” as used herein to describe a mini-chromosome meansthat when an exogenous nucleic acid is present within themini-chromosome the exogenous nucleic acid can function in a detectablemanner when the mini-chromosome is within a plant cell; exemplaryfunctions of the exogenous nucleic acid include transcription of theexogenous nucleic acid, expression of the exogenous nucleic acid,regulatory control of expression of other exogenous nucleic acids,recognition by a restriction enzyme or other endonuclease, ribozyme orrecombinase; providing a substrate for DNA methylation. DNA glycolationor other DNA chemical modification; binding to proteins such ashistones, helix-loop-helix proteins, zinc binding proteins, leucinezipper proteins, MADS box proteins, topoisomerases, helicases,transposases, TATA box binding proteins, viral protein, reversetranscriptases, or cohesins; providing an integration site forhomologous recombination; providing an integration site for atransposon, T-DNA or retrovirus; providing a substrate for RNAisynthesis; priming of DNA replication; aptamer binding; or kinetochorebinding. If multiple exogenous nucleic acids are present within themini-chromosome, the function of one or more of the exogenous nucleicacids can be detected under suitable conditions permitting functionthereof. An “isolated polynucleotide” or “isolated nucleic acid” (andsimilar terms) can refer to a nucleotide sequence (e.g., DNA or RNA)that is not immediately contiguous with nucleotide sequences with whichit is immediately contiguous (one on the 5′ end and one on the 3′ end)in the naturally occurring genome of the organism from which it isderived. Thus, in one embodiment, an isolated nucleic acid includes someor all of the 5′ non-coding (e.g., promoter) sequences that areimmediately contiguous to a coding sequence. The term “isolated” canrefer to a polynucleotide or nucleic acid that is substantially free ofcellular material, viral material, and/or culture medium (when producedby recombinant DNA techniques), or chemical precursors or otherchemicals (when chemically synthesized). “Isolated” does not necessarilymean that the preparation is technically pure (homogeneous), but it issufficiently pure to provide the polynucleotide or nucleic acid in aform in which it can be used for the intended purpose. In certainembodiments, the isolated polynucleotide or nucleic acid is at leastabout 50% pure, e.g., at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, or 99% or more pure.

An “isolated” cell refers to a cell that is at least partially separatedfrom other components with which it is normally associated in itsnatural state. For example, an isolated cell can be a cell in culturemedium.

As used herein, a “library” is a pool of cloned DNA fragments thatrepresents some or all DNA sequences collected, prepared or purifiedfrom a specific source. Each library may contain the DNA of a givenorganism inserted as discrete restriction enzyme generated fragments oras randomly sheared fragments into many thousands of plasmid vectors.For purposes of the present invention, E. coli, yeast, and Salmonellaplasmids are particularly useful for propagating the genome inserts fromother organisms. In principle, any gene or sequence present in thestarting DNA preparation can be isolated by screening the library with aspecific hybridization probe (see, for example, Young et al., In:Eukaryotic Genetic Systems ICN-UCLA Symposia on Molecular and CellularBiology, VII, 315-331, 1977).

As used herein, the term “linker” refers to a DNA molecule, generally upto 50 or 60 nucleotides long and composed of two or more complementaryoligonucleotides that have been synthesized chemically, or excised oramplified from existing plasmids or vectors. In a representativeembodiment, this fragment contains one, or more than one, restrictionenzyme site for a blunt cutting enzyme and/or a staggered cuttingenzyme, such as BamHI. One end of the linker is designed to be ligatableto one end of a linear DNA molecule and the other end is designed to beligatable to the other end of the linear molecule, or both ends may bedesigned to be ligatable to both ends of the linear DNA molecule.

As used herein, a “mini-chromosome” is a recombinant DNA constructincluding a centromere that is capable of transmission to daughtercells. A mini-chromosome may remain separate from the host genome (asepisomes) or may integrate into host chromosomes. The stability of thisconstruct through cell division could range between from about 1% toabout 100%, including about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% and about 95%. The mini-chromosome construct may be a circular orlinear molecule. It may include elements such as one or more telomeres,origin of replication sequences, stuffer sequences, buffer sequences,chromatin packaging sequences, linkers and genes. The number of suchsequences included is only limited by the physical size limitations ofthe construct itself. It could contain DNA derived from a naturalcentromere, although it may be preferable to limit the amount of DNAfrom the natural centromere to the minimal amount required to obtain atransmission efficiency in the range of 1-100%. The mini-chromosomecould also contain a synthetic centromere composed of tandem arrays ofrepeats of any sequence, either derived from a natural centromere, or ofsynthetic DNA. The mini-chromosome could also contain DNA derived frommultiple natural centromeres. The mini-chromosome may be inheritedthrough mitosis or meiosis, or through both meiosis and mitosis. As usedherein, the term mini-chromosome specifically encompasses and includesthe terms “plant artificial chromosome” or “PLAC,” or engineeredchromosomes or microchromosomes and all teachings relevant to a PLAC orplant artificial chromosome specifically apply to constructs within themeaning of the term mini-chromosome.

The term “non-protein expressing sequence” or “non-protein codingsequence” is defined herein as a nucleic acid sequence that is noteventually translated into protein. The nucleic acid may or may not betranscribed into RNA. Exemplary sequences include ribozymes or antisenseRNA.

As used herein, “nucleic acid,” “nucleotide sequence,” and“polynucleotide” are used interchangeably and encompass both RNA andDNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemicallysynthesized) DNA or RNA and chimeras of RNA and DNA. The termpolynucleotide, nucleotide sequence, or nucleic acid refers to a chainof nucleotides without regard to length of the chain. The nucleic acidcan be double-stranded or single-stranded. Where single-stranded, thenucleic acid can be a sense strand or an antisense strand. The nucleicacid can be synthesized using oligonucleotide analogs or derivatives(e.g., inosine or phosphorothioate nucleotides). Such oligonucleotidescan be used, for example, to prepare nucleic acids that have alteredbase-pairing abilities or increased resistance to nucleases. The presentinvention further provides a nucleic acid that is the complement (whichcan be either a full complement or a partial complement) of a nucleicacid, nucleotide sequence, or polynucleotide of the invention.

The term “operably linked” is defined herein as a configuration in whicha control sequence, e.g., a promoter sequence, directs transcription ortranslation of another sequence, for example a coding sequence. Forexample, a promoter sequence could be appropriately placed at a positionrelative to a coding sequence such that the control sequence directs theproduction of a polypeptide encoded by the coding sequence.

“Phenotype” or “phenotypic trait(s)”, as used herein, refers to anobservable property or set of properties resulting from the expressionof a gene. The set of properties may be observed visually or afterbiological or biochemical testing, and may be constantly present or mayonly manifest upon challenge with the appropriate stimulus or activationwith the appropriate signal.

The term “recombinant chromosome” refers to an engineered or artificialchromosome that has been constructed by fragmenting a natural chromosomeand identifying fragmentation products that are capable of segregationthrough mitotic and/or meiotic cell divisions. Recombinant chromosomesare distinct from mini-chromosomes in that they are not constructed invitro from constituent parts and have not been passaged through anheterologous cell such as a bacteria or fungus (as is commonly used instandard cloning techniques). Recombinant chromosomes may the used astargets for addition of transgene expression cassettes.

The term “Sugarcane” refers to any species or hybrid of the genusSaccharum including but not limited to: S. acinaciforme, S. aegyptiacum,S. alopecuroides (Silver Plume Grass), S. alopecuroideum, S.alopecuroidum (Silver Plumegrass), S. alopecurus, S. angustifolium, S.antillarum, S. arenicola, S. argenteum, S. arundinaceum (Hardy SugarCane (Usa)), S. arundinaceum var. trichophyllum, S. asper, S. asperum,S. atrorubens, S. aureum, S. balansae, S. baldwini, S. baldwinii (NarrowPlumegrass), S. barberi (Cultivated Sugarcane), S. barbicoatatum, S.beccarii, S. bengalense (Munj Sweetcane), S. benghalense, S. bicorne, S.bflorum, S. boga, S. brachypogon, S. bructealum, S. brasiliamum, S.brevibarbe (Short-Beard Plume Grass), S. brevibarbe var. brevibarbe(Shortbeard Plumegrass), S. brevibarbe var. contortum (ShortbeardPlumegrass), S. brevifolium, S. brunneum, S. caducum, S. canaliculatum,S. capense, S. casi, S. caudatum, S. cayennense, S. cayennene var.genuinum, S. cayennense var. laxiusculum, S. chinense, S. ciliare, S.coarclatum (Compressed Plumegrass), S. confertum, S. conjugatum, S.contortum, S. contortum var. contortum, S. contractum, S. cotuliferum,S. cylindricum, S. cylindricum var. contractum, S. cylindricum var.longifolium, S. deciduum, S. densum, S. diandrum, S. dissitiflorum, S.distichophyllum, S. dublum, S. ecklonli, S. edule, S. elegans, S.elephantinum, S. erlantholdes, S. europaeum, S. exaltatum, S.fasciculalum, S. fasllgiatum, S. fatuum, S. fllifolium, S. filiforme, S.floridulum, S. formosanum, S. fragile, S. fidulvum, S. fuscum, S.giganteum (Sugarcane Plume Grass), S. glabrum, S. glaga, S. glaucum, S.glaza, S. grandiflorum, S. griffithii, S. hildebrandtii, S. hirsulum, S.holcoides, S. holcoides var. warmingianum, S. hookeri, S. hybrid, S.hybridum, S. indum, S. infirmum, S. insulare, S. irritans, S.jaculalorium, S. jamaicense, S. japonicum, S. juncifolium, S.kajkalense, S. kanashiroi, S. klagha, S. koenigii, S. laguroides, S.longifolium, S. longisetosum, S. longiretosum var. hookeri, S.longisetum, S. lota, S. luzonicum, S. macilentum, S. macrantherum, S.maximum, S. mexicanum, S. modhara, S. monandrum, S. moonja, S. munja, S.munroanum, S. muticum, S. narenga (Narenga Sugarcane), S. negrosense, S.obscurum, S. occidentale, S. officinale, S. oficinalis, S. officinarum(Cultivated Sugarcane), S. officinarum ‘Choribon’, S. oficinarum‘Otaheite’, S. officinarum ‘Pele's Smoke’ (Black Magic Repellent Plant),S. officinarum L. ‘Laukona’, S. officinarum L. ‘Violaceum’, S.officinarum var. brevipedicellatum, S. officinarum var. oficinarum, S.officinarum var. violaceum (Burgundy-Leaved Sugarcane), S. pallidum, S.paniceum, S. panicosum, S. pappiferum, S. parviflorum, S. pedicellare,S. perrieri, S. polydactylum, S. polystachyon, S. polystachyum, S.purphyrocomum, S. procerum, S. propinquum, S. punctatum, S. rara, S.rarum, S. ravennae (Hardy Pampas Plume Grass), S. repens, S. reptans, S.ridleyi, S. robustum (Wild New-Guinean Cane), S. roseum, S. rubicundum,S. rufum, S. sagittatum, S. sanguineum, S. sape, S. sara, S. scindicus,S. semidecumbens, S. sibiricum, S. sikkimense, S. sinense (CultivatedSugarcane), S. sisca, S. sorghum, S. speciosissimum, S. sphacelatum, S.spicatum, S. spontaneum (Wild Sugar Cane), S. spontaneum var. insulare,S. spontanum, S. stenophyllum, S. stewartii, S. strictum, S. tenerijfae,S. ternatum, S. thunbergii, S. tinctorium, S. tridentatum, S. trinii, S.tristachyum, S. velutinum, S. versicolor, S. viguieri, S. villosum, S.violaceum, S. wardii, S. warminglanum, S. williamsii.

As used herein, the term “Sugarcane Basic Mini-Chromosome” is defined asa recombinant DNA construct that when present within a Sugarcane cell iscapable of mitotic and/or meiotic transmission to Sugarcane daughtercells under appropriate conditions and comprises a Sugarcane AssembledCentromere and, optionally, one or more of the following:

-   -   (a) one or more telomeres;    -   (b) one or more sequences for regulating, maintaining, or        imparting topological or chromatin structure, molecular        integrity, or stability of gene expression or inheritance in        Sugarcane;    -   (c) the required vector DNA that allows for propagation of the        mini-chromosome in Sugarcane and DNA that facilitates the        selective removal of unwanted portions of the mini-chromosome        prior to or after Sugarcane transformation; or    -   (d) a Transgene Expression Cassette, wherein the Transgene        Expression Cassette serves only to regulate, maintain, or impart        function or stability to a mini-chromosome in Sugarcane.

A “Sugarcane Basic Mini-Chromosome” does not include a SugarcaneTransgene Expression Cassette that imparts one or more functions otherthan those expressly set forth in subsection (d), above.

As used herein, a “Sugarcane Assembled Centromere” means apolynucleotide sequence having the properties of a Centromere that isassembled from one or more fragments of native Centromere(s) and/orother polynucleotide sequence, which are (i) isolated from a plant cell,and/or based on plant Centromere sequence motifs, (ii) inserted into avector (e.g. a plasmid vector) that is propagated and maintained in acell of a heterologous organism, and (iii) delivered back into aSugarcane plant cell as part of a Sugarcane Basic or Sugarcane AppliedMini-Chromosome. A Sugarcane Assembled Centromere may possibly bemodified by an endogenous in vivo process after it is delivered into aSugarcane plant cell such that its sequence now differs from thatcontained in the parental Sugarcane Basic or Sugarcane AppliedMini-Chromosome as propagated in a cell of a heterologous organism. Forthe avoidance of doubt a Sugarcane Assembled Centromere does not includederivatives or deletions of native Sugarcane Centromeres that areconstructed within the Sugarcane plant cell, and are never maintained intheir entirety in a cell of a heterologous organism.

As used herein, a “Sugarcane Applied Mini-Chromosome” means a geneticconstruct formed by integrating one or more Transgene ExpressionCassettes into a Sugarcane Basic Mini-Chromosome, wherein said TransgeneExpression Cassettes impart one or more functions other than toregulate, maintain, or impart function or stability to amini-chromosome.

The term “plant part” as used herein includes a pod, root, sett root,shoot root, root primordial, shoot, primary shoot, secondary shoot,tassle, panicle, arrow, midrib, blade, ligule, auricle, dewlap, bladejoint, sheath, node, internode, bud furrow, leaf scar, cutting, tuber,stem, stalk, fruit, berry, nut, flower, leaf, bark, wood, epidermis,vascular tissue, organ, protoplast, crown, callus culture, petiole,petal, sepal, stamen, stigma, style, bud, meristem, cambium, cortex,pith, sheath, silk, ovule or embryo. Other exemplary Sugarcane plantparts are a meiocyte or gamete or ovule or pollen or endosperm of any ofthe plants of the invention. Other exemplary plant parts are a seed,seed-piece, embryo, protoplast, cell culture, any group of plant cellsorganized into a structural and functional unit, ratoon or propagule.

The term “promoter” is defined herein as a DNA sequence that allows thebinding of RNA polymerase (including but not limited to RNA polymeraseI, RNA polymerase II and RNA polymerase III from eukaryotes) and directsthe polymerase to a downstream transcriptional start site of a nucleicacid sequence encoding a polypeptide to initiate transcription. RNApolymerase effectively catalyzes the assembly of messenger RNAcomplementary to the appropriate DNA strand of the coding region.

A “promoter operably linked to a heterologous gene” is a promoter thatis operably linked to a gene that is different from the gene to whichthe promoter is normally operably linked in its native state. Similarly,an “exogenous nucleic acid operably linked to a heterologous regulatorysequence” is a nucleic acid that is operably linked to a regulatorycontrol sequence to which it is not normally linked in its native state.

The term “hybrid promoter” is defined herein as parts of two or morepromoters that are fused together to generate a sequence that is afusion of the two or more promoters, which is operably linked to acoding sequence and mediates the transcription of the coding sequenceinto mRNA.

The term “tandem promoter” is defined herein as two or more promotersequences each of which is operably linked to a coding sequence andmediates the transcription of the coding sequence into mRNA.

The term “constitutive active promoter” is defined herein as a promoterthat allows permanent stable expression of the gene of interest.

The term “Inducible promoter” is defined herein as a promoter induced bythe presence or absence of a biotic or an abiotic factor.

The term “polypeptide” does not refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “exogenous polypeptide” is defined as a polypeptidewhich is not native to the plant cell, a native polypeptide in whichmodifications have been made to alter the native sequence, or a nativepolypeptide whose expression is quantitatively altered as a result of amanipulation of the plant cell by recombinant DNA techniques.

As used herein, the term “pseudogene” refers to a non-functional copy ofa protein-coding gene; pseudogenes found in the genomes of eukaryoticorganisms are often inactivated by mutations and are thus presumed to benon-essential to that organism; pseudogenes of reverse transcriptase andother open reading frames found in retroelements are abundant in thecentromeric regions of Arabidopsis and other organisms and are oftenpresent in complex clusters of related sequences.

As used herein the term “regulatory sequence” refers to any DNA sequencethat influences the efficiency of transcription or translation of anygene. The term includes, but is not limited to, sequences comprisingpromoters, enhancers and terminators.

As used herein the term “repeated nucleotide sequence” refers to anynucleic acid sequence of at least 25 bp present in a genome or arecombinant molecule, other than a telomere repeat, that occurs at leasttwo or more times and that are optionally at least 80% identical eitherin head to tail or head to head orientation either with or withoutintervening sequence between repeat units.

As used herein, the term “retroelement” or “retrotransposon” refers to agenetic element related to retroviruses that disperse through an RNAstage; the abundant retroelements present in plant genomes contain longterminal repeats (LTR retrotransposons) and encode a polyprotein genethat is processed into several proteins including a reversetranscriptase. Specific retroelements (complete or partial sequences)can be found in and around plant centromeres and can be present asdispersed copies or complex repeat clusters. Individual copies ofretroelements may be truncated or contain mutations; intactretroelements are rarely encountered.

As used herein the term “satellite DNA” refers to short DNA sequences(typically <1000 bp) present in a genome as multiple repeats, mostlyarranged in a tandemly repeated fashion, as opposed to a dispersedfashion. Repetitive arrays of specific satellite repeats are abundant inthe centromeres of many higher eukaryotic organisms.

As used herein, a “screenable marker” is a gene whose presence resultsin an identifiable phenotype. This phenotype may be observable understandard conditions, altered conditions such as elevated temperature, orin the presence of certain chemicals used to detect the phenotype. Theuse of a screenable marker allows for the use of lower, sub-killingantibiotic concentrations and the use of a visible marker gene toidentify clusters of transformed cells, and then manipulation of thesecells to homogeneity. Illustrative screenable markers of the presentinclude genes that encode fluorescent proteins that are detectable by avisual microscope such as the fluorescent reporter genes DsRed, ZsGren,ZsYellow, AmCyan, Green Fluorescent Protein (GFP) and modifications ofthese reporter genes to excite or emit at altered wavelengths. Anadditional screenable marker gene is lac.

Alternative methods of screening for modified plant cells may involveuse of relatively low, sub-killing concentrations of a selection agent(e.g. sub-killing antibiotic concentrations), and also involve use of ascreenable marker (e.g., a visible marker gene) to identify clusters ofmodified cells carrying the screenable marker, after which thesescreenable cells are manipulated to homogeneity. As used herein, a“selectable marker” is a gene whose presence results in a clearphenotype, and most often a growth advantage for cells that contain themarker. This growth advantage may be present under standard conditions,altered conditions such as elevated temperature, specialized mediacompositions, or in the presence of certain chemicals such as herbicidesor antibiotics. Use of selectable markers is described, for example, inBroach et al. Gene, 8:121-133, 1979. Examples of selectable markersinclude the thymidine kinase gene, the cellular adeninephosphoribosyltransferase gene and the dihydrylfolate reductase gene,hygromycin phosphotransferase genes, the bar gene, neomycinphosphotransferase genes and phosphomannose isomerase gene, amongothers. Nonlimiting examples of selectable markers in the presentinvention include genes whose expression confer antibiotic or herbicideresistance to the host cell, or proteins allowing utilization of acarbon source not normally utilized by plant cells. Expression of one ofthese markers should be sufficient to enable the survival of those cellsthat comprise a vector within the host cell, and facilitate themanipulation of the vector into new host cells. Of particular interestin the present invention are proteins conferring cellular resistance tokanamycin, G 418, paramomycin, hygromycin, bialaphos, and glyphosate forexample, or proteins allowing utilization of a carbon source, such asmannose, not normally utilized by plant cells.

The term “stable” as used herein means that the mini-chromosome can betransmitted to daughter cells over at least 8 mitotic generations. Someembodiments of mini-chromosomes may be transmitted as functional,autonomous units for less than 8 mitotic generations, e.g. 1, 2, 3, 4,5, 6, or 7. According to representative embodiments, the mini-chromosomecan be transmitted over at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 generations,for example, through the regeneration or differentiation of an entireplant, and may even be are transmitted through meiotic division togametes. Other representative mini-chromosomes can be further maintainedin the zygote derived from such a gamete or in an embryo or endospermderived from one or more such gametes. A “functional and stable”mini-chromosome is one in which functional mini-chromosomes can bedetected after transmission of the mini-chromosomes over at least 8mitotic generations, or after inheritance through a meiotic division.During mitotic division, as occurs occasionally with native chromosomes,there may be some non-transmission of mini-chromosomes; themini-chromosome may still be characterized as stable despite theoccurrence of such events if an adchromosomal plant that containsdescendants of the mini-chromosome distributed throughout its parts maybe regenerated from cells, cuttings, propagules, or cell culturescontaining the mini-chromosome, or if an adchromosomal plant can beidentified in progeny of the plant containing the mini-chromosome.

As used herein, a “structural gene” is a sequence which codes for apolypeptide or RNA and includes 5′ and 3′ ends. The structural gene maybe from the host into which the structural gene is transformed or fromanother species. A structural gene may optionally, but not necessarily,include one or more regulatory sequences which modulate the expressionof the structural gene, such as a promoter, terminator or enhancer. Astructural gene may optionally, but not necessarily, confer some usefulphenotype upon an organism comprising the structural gene, for example,herbicide resistance. In one embodiment of the invention, a structuralgene may encode an RNA sequence which is not translated into a protein,for example a tRNA or rRNA gene.

As used herein, the term “telomere” or “telomere DNA” refers to asequence capable of capping the ends of a chromosome, thereby preventingdegradation of the chromosome end, ensuring replication and preventingfusion to other chromosome sequences. Telomeres can include naturallyoccurring telomere sequences or synthetic sequences. Telomeres from onespecies may confer telomere activity in another species. An exemplarytelomere DNA is a heptanucleotide telomere repeat TITAGGG (and itscomplement) found in the majority of plants.

“Transformed,” “transgenic,” “modified,” and “recombinant” refer to ahost organism such as a plant into which an exogenous or heterologousnucleic acid molecule has been introduced, and includes meiocytes,seeds, zygotes, embryos, endosperm, or progeny of such plant that retainthe exogenous or heterologous nucleic acid molecule but which have notthemselves been subjected to the transformation process.

When the phrase “transmission efficiency” of a certain percent is used,transmission percent efficiency is calculated by measuringmini-chromosome presence through one or more mitotic or meioticgenerations. It is directly measured as the ratio (expressed as apercentage) of the daughter cells or plants demonstrating presence ofthe mini-chromosome to parental cells or plants demonstrating presenceof the mini-chromosome. Presence of the mini-chromosome in parental anddaughter cells can be demonstrated with assays that detect the presenceof an exogenous nucleic acid carried on the mini-chromosome. Exemplaryassays can be the detection of a screenable marker (e.g. presence of afluorescent protein or any gene whose expression results in anobservable phenotype), a selectable marker, or PCR amplification of anyexogenous nucleic acid carried on the mini-chromosome.

Constructing Mini-Chromosomes by Site-Specific Recombination

Sugarcane mini-chromosomes may be constructed using site-specificrecombination sequences (for example those recognized by thebacteriophage P1 Cre recombinase, or the bacteriophage lambda integrase,or similar recombination enzymes). According to this embodiment, acompatible recombination site, or a pair of such sites, is present onboth the Sugarcane centromere containing DNA clones and the donor DNAclones. Incubation of the donor clone and the centromere clone in thepresence of the recombinase enzyme causes strand exchange to occurbetween the recombination sites in the two plasmids; the resultingSugarcane mini-chromosomes contain Sugarcane centromere sequences aswell as mini-chromosome vector sequences. The DNA molecules formed insuch recombination reactions are introduced into E. coli, otherbacteria, yeast or Sugarcane cells by common methods in the fieldincluding, but not limited to, heat shock, chemical transformation,electroporation, particle bombardment, whiskers, or other transformationmethods followed by selection for marker genes including chemical,enzymatic, color, or other marker, allowing for the selection oftransformants harboring mini-chromosomes.

II. Methods of Detecting and Characterizing Mini-Chromosomes in PlantCells or of Scoring Mini-Chromosome Performance in Plant Cells:Identification of Candidate Centromere Fragments by Probing BACLibraries

Sugarcane centromere clones are identified from a large Sugarcanegenomic insert library such as a Bacterial Artificial Chromosomelibrary. Probes are labeled using nick-translation in the presence ofradioactively labeled dCTP, dATP, dGTP or dTTP as in, for example, thecommercially available Rediprime kit (Amersham) as per themanufacturer's instructions. Other labeling methods familiar to thoseskilled in the art could be substituted. The libraries are screened anddeconvoluted. Sugarcane genomic clones are screened by probing withsmall centromere-specific clones. Other embodiments of this procedurewould involve hybridizing a library with other centromere sequences. Ofthe BAC clones identified using this procedure, a representative set areidentified as having high hybridization signals to some probes, andoptionally low hybridization signals to other probes. These areselected, the bacterial clones grown up in cultures and DNA prepared bymethods familiar to those skilled in the art such as alkaline lysis. TheDNA composition of purified clones is surveyed using for examplefingerprinting by digesting with restriction enzymes such as, but notlimited to, HinfI or HindIII. In a representative embodiment therestriction enzyme cuts within the tandem centromere satellite repeat(see below). A variety of clones showing different fingerprints areselected for conversion into mini-chromosomes and inheritance testing.It can also be informative to use multiple restriction enzymes forfingerprinting or other enzymes which can cleave DNA.

Fingerprinting Analysis of BACs and Mini-Chromosomes

Sugarcane centromere function may be associated with large tandem arraysof satellite repeats. To assess the composition and architecture of thecentromere BACs, the candidate BACs are digested with a restrictionenzyme, such as HindII, which cuts with known frequency within theconsensus sequence of the unit repeat of the tandemly repeatedcentromere satellite. Digestion products are then separated by agarosegel electrophoresis. Large insert clones containing a large array oftandem repeats will produce a strong band of the unit repeat size, aswell as less intense bands at 2× and 3× the unit repeat size, andfurther multiples of the repeat size. These methods are well-known andthere are many possible variations known to those skilled in the art.

Determining Sequence Composition of Mini-Chromosomes by ShotgunCloning/Sequencing, Sequence Analysis

To determine the sequence composition of the Sugarcane mini-chromosome,the centromeric region of the Sugarcane mini-chromosome is sequenced. Togenerate DNA suitable for sequencing. Sugarcane mini-chromosomes arefragmented, for example by using a random shearing method (such assonication, nebulization, etc). Other fragmentation techniques may alsobe used such as enzymatic digestion. These fragments are then clonedinto a vector (e.g., a plasmid) and sequenced. The resulting DNAsequence is trimmed of poor-quality sequence and of sequencecorresponding to the vector. The sequence is then compared to the knownDNA sequences using an algorithm such as BLAST to search a sequencedatabase such as GenBank.

To determine the consensus of the Sugarcane satellite repeat in theSugarcane mini-chromosome, the sequences containing the satellite repeatare aligned using a DNA sequence alignment program such as ContigExpressfrom Vector NTI. The sequences may also be aligned to previouslydetermined repeats for that species. The sequences are trimmed to unitrepeat length using the consensus as a template. Sequences trimmed fromthe ends of the alignment are realigned with the consensus and furthertrimmed until all sequences are at or below the consensus length. Thesequences are then aligned with each other. The consensus is determinedby the frequency of a specific nucleotide at each position; if the mostfrequent base is three times more frequent than the next most frequentbase, it was considered the consensus.

Methods for determining consensus sequence are well known in the art,see, e.g., U.S. Pat. App. Pub. No. 20030124561; Hall & Preuss (2002).These methods, including DNA sequencing, assembly, and analysis, arewell-known and there are many possible variations known to those skilledin the art. Other alignment parameters may also be useful such as usingmore or less stringent definitions of consensus.

Non-Selective Mini-Chromosome Mitotic Inheritance Assays

The following list of assays and potential outcomes illustrates howvarious assays can be used to distinguish autonomous events fromintegrated events.

Assay #1: Transient Assay

Sugarcane mini-chromosomes are tested for their ability to becomeestablished as chromosomes and their ability to be inherited in mitoticcell divisions. In this assay, Sugarcane mini-chromosomes are deliveredto Sugarcane plant cells, for example suspension cells in liquidculture. The cells used can be at various stages of growth. Optionally,a population in which some cells are undergoing division can be used.The Sugarcane mini-chromosome is then assessed over the course ofseveral cell divisions, by tracking the presence of a screenable marker,e.g. a visible marker gene such as a fluorescent protein. Sugarcanemini-chromosomes that are established and inherited well may show aninitial delivery into many single cells; after several cell divisions,these single cells divide to form clusters of mini-chromosome-containingcells. Other exemplary embodiments of this method include deliveringSugarcane mini-chromosomes to other mitotic cell types, including rootsand shoot meristems.

Assay #2: Non-Lineage Based Inheritance Assays on Modified TransformedCells and Plants

Sugarcane mini-chromosome inheritance is assessed on modified cell linesand plants by following the presence of the mini-chromosome over thecourse of multiple cell divisions. An initial population of Sugarcanemini-chromosome containing cells is assayed for the presence of theSugarcane mini-chromosome, by the presence of a marker gene, includingbut not limited to a fluorescent protein, a colored protein, a proteinassayable by histochemical assay, and a gene affecting cell morphology.In the use of a DNA-specific dye, all nuclei are stained with a dyeincluding but not limited to DAPI, Hoechst 33258, OliGreen, Giemsa YOYO,or TOTO, allowing a determination of the number of cells that do notcontain the mini-chromosome. After the initial determination of thepercent of cells carrying the Sugarcane mini-chromosome, the remainingcells are allowed to divide over the course of several cell divisions.The number of cell divisions, n, is determined by a method including butnot limited to monitoring the change in total weight of cells, andmonitoring the change in volume of the cells or by directly countingcells in an aliquot of the culture. After a number of cell divisions,the population of cells is again assayed for the presence of theSugarcane mini-chromosome. The loss rate per generation is calculated bythe equation:

Loss rate per generation=1−(F/I)^(1/n)

The population of Sugarcane mini-chromosome-containing cells may includesuspension cells, callus, roots, leaves, meristems, flowers, or anyother tissue of modified plants, or any other cell type containing amini-chromosome.

These methods are well-known and there are many possible variationsknown to those skilled in the art; they have been used before with humancells and yeast cells.

Assay #3: Lineage Based Inheritance Assays on Modified Cells and Plants

Sugarcane mini-chromosome inheritance is assessed on cell lines andplants comprising Sugarcane mini-chromosomes by following the presenceof the Sugarcane mini-chromosome over the course of multiple celldivisions. In cell types that allow for tracking of cell lineage,including but not limited to root or leaf cell files, trichomes, andleaf stomata guard cells, Sugarcane mini-chromosome loss per generationdoes not need to be determined statistically over a population, it canbe discerned directly through successive cell divisions. In othermanifestations of this method, cell lineage can be discerned from cellposition, or methods including but not limited to the use ofhistological lineage tracing dyes, and the induction of genetic mosaicsin dividing cells.

In one simple example, the two guard cells of the stomata are daughtersof a single precursor cell. To assay Sugarcane mini-chromosomeinheritance in this cell type, the epidermis of the leaf of a Sugarcaneplant containing a Sugarcane mini-chromosome is examined for thepresence of the Sugarcane mini-chromosome by the presence of a markergene, including but not limited to a fluorescent protein, a coloredprotein, a protein assayable by histochemical assay, and a geneaffecting cell morphology. The number of loss events in which one guardcell contains the Sugarcane mini-chromosome (L) and the number of celldivisions in which both guard cells contain the Sugarcanemini-chromosome (B) are counted. The loss rate per cell division isdetermined as L/(L+B). Other lineage-based cell types are assayed insimilar fashion. These methods are well-known and there are manypossible variations known to those skilled in the art; they have beenused before with yeast cells (though, instead of observing the marker instomates, a color marker was observed in yeast colonies).

Linear Sugarcane mini-chromosome inheritance may also be assessed byexamining leaf or root files or clustered cells in callus over time.Changes in the percent of cells carrying the Sugarcane mini-chromosomewill indicate the mitotic inheritance.

Assay #4: Inheritance Assays on Modified Cells and Plants in thePresence of Chromosome Loss Agents

Any of the above three assays can be done in the presence of chromosomeloss agents (including but not limited to colchicine, colcemid,caffeine, etopocide, nocodazole, oryzalin, trifluran). It is likely thatan autonomous Sugarcane mini-chromosome will prove more susceptible toloss induced by chromosome loss agents; therefore, autonomousmini-chromosomes should show a lower rate of inheritance in the presenceof chromosome loss agents. These methods have been used to studychromosome loss in fruit flies and yeast; there are many possiblevariations known to those skilled in the art.

III. Transformation of Plant Cells and Plant Regeneration

Various methods may be used to deliver DNA into plant cells. Theseinclude biological methods, such as Agrobacterium, E. coli, and viruses,physical methods such as biolistic particle bombardment, nanocopoeadevice, the Stein beam gun, silicon carbide whiskers and microinjection,electrical methods such as electroporation, and chemical methods such asthe use of poly-ethylene glycol and other compounds known to stimulateDNA uptake into cells. Examples of these techniques are described byPaszkowski et al., EMBO J 3: 2717-2722 (1984), Potrykus et al., Mol.Gen. Genet. 199: 169-177 (1985), Reich et al., Biotechnology 4:1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987).Transformation using silicon carbide whiskers, e.g. in maize, isdescribed in Brisibe, J. Exp. Bot. 51(343):187-196 (2000) and Dunwell,Methods Mol. Biol. 111:375-82 (1999) and U.S. Pat. No. 5,464,765.

Agrobacterium-Mediated Delivery

Agrobacterium-mediated transformation is one method for introducing adesired genetic element into a plant. Several Agrobacterium speciesmediate the transfer of a specific DNA known as “T-DNA” that can begenetically engineered to carry a desired piece of DNA into many plantspecies. Plasmids used for delivery contain the T-DNA flanking thenucleic acid to be inserted into the plant. The major events marking theprocess of T-DNA mediated pathogenesis are induction of virulence genes,processing and transfer of T-DNA.

There are three common methods to transform plant cells withAgrobacterium. The first method is co-cultivation of Agrobacterium withcultured isolated protoplasts. This method requires an establishedculture system that allows culturing protoplasts and plant regenerationfrom cultured protoplasts. The second method is transformation of cellsor tissues with Agrobacterium. This method requires (a) that the plantcells or tissues can be modified by Agrobacterium and (b) that themodified cells or tissues can be induced to regenerate into wholeplants. The third method is transformation of seeds, immature or matureembryos, apices or meristems with Agrobacterium. This method requiresexposure of the meristematic cells of these tissues to Agrobacterium andmicropropagation of the shoots or plant organs arising from thosemeristematic cells.

Those of skill in the art are familiar with procedures for growth andsuitable culture conditions for Agrobacterium as well as subsequentinoculation procedures. Liquid, solid, or semi-solid culture media canbe used. The density of the Agrobacterium culture used for inoculationand the ratio of Agrobacterium cells to explant can vary from one systemto the next, as can media, growth procedures, timing and lightingconditions.

Transformation of dicotyledons using Agrobacterium has long been knownin the art, and transformation of monocotyledons using Agrobacterium hasalso been described. See, WO 94/00977 and U.S. Pat. No. 5,591,616, bothof which are incorporated herein by reference. See also, Negrotto etal., Plant Cell Reports 19: 798-803 (2000), incorporated herein byreference.

A number of wild-type and disarmed strains of Agrobacterium tumefaciensand Agrobacterium rhizogenes harboring Ti or Ri plasmids can be used forgene transfer into plants. In embodiments of the invention, theAgrobacterium hosts contain disarmed Ti and Ri plasmids that do notcontain the oncogenes that cause tumorigenesis or rhizogenesis.Exemplary strains include Agrobacterium lumefaciens strain C58, anopaline-type strain that is used to mediate the transfer of DNA into aplant cell, octopine-type strains such as LBA4404 or succinamopine-typestrains, e.g., EHA101 or EHA105. The use of these strains for planttransformation has been reported and the methods are familiar to thoseof skill in the art.

U.S. Application No. 20040244075 published Dec. 2, 2004 describesimproved methods of Agrobacterium-mediated transformation. Theefficiency of transformation by Agrobacterium may be enhanced by using anumber of methods known in the art. For example, the inclusion of anatural wound response molecule such as acetosyringone (AS) to theAgrobacterium culture has been shown to enhance transformationefficiency with Agrobacterium tumefaciens (Shahla et al., (1987) PlantMolec. Biol. 8:291-298). Alternatively, transformation efficiency may beenhanced by wounding the target tissue to be modified or transformed.Wounding of plant tissue may be achieved, for example, by punching,maceration, bombardment with microprojectiles, etc. (See e.g., Bidney etal., (1992) Plant Molec. Biol. 18:301-313).

In addition, another recent method described by Broothaerts, et. al.(Nature 433: 629-633, 2005) expands the bacterial genera that can beused to transfer genes into plants. This work involved the transfer of adisarmed Ti plasmid without T-DNA and another vector with T-DNAcontaining the marker enzyme beta-glucuronidase, into three differentbacteria. Gene transfer was successful and this method significantlyexpands the tools available for gene delivery into plants.

Microprojectile Bombardment Delivery

Another widely used technique to genetically transform plants involvesthe use of microprojectile bombardment. In this process, a nucleic acidcontaining the desired genetic elements to be introduced into the plantis deposited on or in small dense particles, e.g., tungsten, platinum,or 0.5 to 1.0 micron gold particles, which are then delivered at a highvelocity into the plant tissue or plant cells using a specializedbiolistics device. Many such devices have been designed and constructed;one in particular, the PDS1000/He sold by BioRad, is the instrument mostcommonly used for biolistics of plant cells. The advantage of thismethod is that no specialized sequences need to be present on thenucleic acid molecule to be delivered into plant cells; delivery of anynucleic acid sequence is theoretically possible.

For the bombardment, cells in suspension are concentrated on filters,petri dishes or solid culture medium. Alternatively, immature embryos,seedling explants, or any plant tissue or target cells may be arrangedon filters, petri dishes or solid culture medium. The cells to bebombarded are positioned at an appropriate distance below themicroprojectile stopping plate.

Various biolistics protocols have been described that differ in the typeof particle or the manner in which DNA is coated onto the particle. Anytechnique for coating microprojectiles that allows for delivery oftransforming DNA to the target cells may be used. For example, particlesmay be prepared by functionalizing the surface of a gold particle byproviding free amine groups. DNA, having a strong negative charge, willthen bind to the functionalized particles.

Parameters such as the concentration of DNA used to coatmicroprojectiles may influence the recovery of transformants containinga single copy of the transgene. For example, a lower concentration ofDNA may not necessarily change the efficiency of the transformation butmay instead increase the proportion of single copy insertion events. Inthis regard, ranges of approximately 1 ng to approximately 10 μg (10,000ng), approximately 5 ng to 8 μg or approximately 20 ng, 50 ng, 100 ng,200 ng, 500 ng, 1 μg, 2 μg, 5 μg, or 7 g of transforming DNA may be usedper each 1.0-2.0 mg of starting gold particles (in the 0.5 to 1.0 micronrange).

Other physical and biological parameters may be varied, such asmanipulation of the DNA/microprojectile precipitate, factors that affectthe flight and velocity of the projectiles, manipulation of the cellsbefore and immediately after bombardment (including osmotic state,tissue hydration and the subculture stage or cell cycle of the recipientcells), the orientation of an immature embryo or other target tissuerelative to the particle trajectory, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.One may also want to use agents to protect the DNA during delivery. Onemay particularly wish to adjust physical parameters such as DNAconcentration, gap distance, flight distance, tissue distance, andhelium pressure.

The particles delivered via biolistics can be “dry” or “wet.” In the“dry” method, the mini-chromosome DNA-coated particles such as gold areapplied onto a macrocarrier (such as a metal plate, or a carrier sheetmade of a fragile material such as mylar) and dried. The gas dischargethen accelerates the macrocarrier into a stopping screen, which haltsthe macrocarrier but allows the particles to pass through; the particlesthen continue their trajectory until they impact the tissue beingbombarded. For the “wet” method, the droplet containing themini-chromosome DNA-coated particles is applied to the bottom part of afilter holder, which is attached to a base which is itself attached to arupture disk holder used to hold the rupture disk to the helium egresstube for bombardment. The gas discharge directly displaces the DNA/golddroplet from the filter holder and accelerates the particles and theirDNA cargo into the tissue being bombarded. The wet biolistics method hasbeen described in detail elsewhere but has not previously been appliedin the context of plants (Mialhe et al., Mol Mar Biol Biotechnol.4(4):275-83, 1995). The concentrations of the various components forcoating particles and the physical parameters for delivery can beoptimized using procedures known in the art. A variety of Sugarcanecells/tissues are suitable for transformation, including immatureembryos, scutellar tissue, suspension cell cultures, immatureinflorescence, shoot meristem, epithelial peels, nodal explants, callustissue, hypocotyl tissue, cotyledons, roots, leaves, meristem cells, andgametic cells such as microspores, pollen, sperm and egg cells. It iscontemplated that any cell from which a fertile plant may be regeneratedis useful as a recipient cell. Callus may be initiated from tissuesources including, but not limited to, immature embryos, seedling apicalmeristems, microspore-derived embryos, roots, hypocotyls, cotyledons andthe like. Those cells which are capable of proliferating as callus alsoare recipient cells for genetic transformation.

Any suitable plant culture medium can be used. Examples of suitablemedia would include but are not limited to MS-based media (Murashige andSkoog, Physiol. Plant, 15:473-497, 1962) or N6-based media (Chu et al.,Scientia Sinica 18:659, 1975) supplemented with additional plant growthregulators including but not limited to auxins such as picloram(4-amino-3,5,6-trichloropicolinic acid), 2,4-D(2,4-dichlorophenoxyacetic acid), naphalene-acetic acid (NAA) anddicamba (3,6-dichloroanisic acid), cytokinins such as BAP(6-benzylaminopurine) and kinetin, and gibberellins. Other mediaadditives can include but are not limited to amino acids, macroelements,iron, microelements, vitamins and organics, carbohydrates, undefinedmedia components such as casein hydrolysates, an appropriate gellingagent such as a form of agar, a low melting point agarose or Gelrite ifdesired. Those of skill in the art are familiar with the variety oftissue culture media, which when supplemented appropriately, supportplant tissue growth and development and are suitable for planttransformation and regeneration. These tissue culture media can eitherbe purchased as a commercial preparation, or custom prepared andmodified. Examples of such media would include but are not limited toMurashige and Skoog (Murashige and Skoog, Physiol. Plant, 15:473-497,1962), N6 (Chu et al., Scientia Sinica 18:659, 1975), Linsmaier andSkoog (Linsmaier and Skoog, Physio. Plant., 18:100, 1965), Uchimiya andMurashige (Uchimiya and Murashige, Plant Physiol. 15:473, 1962),Gamborg's B5 media (Gamborg et al., Exp. Cell Res., 50:151, 1968), Dmedium (Duncan et al., Planta, 165:322-332, 1985), Mc-Cown's Woody plantmedia (McCown and Lloyd, HortScience 6:453, 1981), Nitsch and Nitsch(Nitsch and Nitsch, Science 163:85-87, 1969), and Schenk and Hildebrandt(Schenk and Hildebrandt, Can. J. Bot. 50:199-204, 1972) or derivationsof these media supplemented accordingly. Those of skill in the art areaware that media and media supplements such as nutrients and growthregulators for use in transformation and regeneration and other cultureconditions such as light intensity during incubation, pH, and incubationtemperatures can be varied.

Those of skill in the art are aware of the numerous modifications inselective regimes, media, and growth conditions that can be varieddepending on the plant system and the selective agent. Typical selectiveagents include but are not limited to antibiotics such as geneticin(G418), kanamycin, paromomycin or other chemicals such as glyphosate orother herbicides. Consequently, such media and culture conditionsdisclosed in the present invention can be modified or substituted withnutritionally equivalent components, or similar processes for selectionand recovery of transgenic events, and still fall within the scope ofthe present invention.

Sugarcane Mini-Chromosome Delivery without Selection

The Sugarcane mini-chromosome is delivered to Sugarcane plant cells ortissues, e.g., plant cells in suspension to obtain stably modifiedcallus clones for inheritance assay. Suspension cells are maintained ina growth media, for example Murashige and Skoog (MS) liquid mediumcontaining an auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D).Cells are bombarded using a particle bombardment process, such as thehelium-driven PDS-1000/He system, and propagated in the same liquidmedium to permit the growth of modified and non-modified cells. Portionsof each bombardment are monitored for formation of fluorescent clusters,which are isolated by micromanipulation and cultured on solid medium.Clones modified with the mini-chromosome are expanded and homogenousclones are used in inheritance assays, or assays measuringmini-chromosome structure or autonomy.

Sugarcane Mini-Chromosome Transformation with Selectable Marker Gene

Isolation of Sugarcane mini-chromosome-modified cells in bombardedcalluses or explants can be facilitated by the use of a selectablemarker gene. The bombarded tissues are transferred to a mediumcontaining an appropriate selective agent for a particular selectablemarker gene. Such a transfer usually occurs between 0 and about 7 daysafter bombardment. The transfer could also take place any number of daysafter bombardment. The amount of selective agent and timing ofincorporation of such an agent in selection medium can be optimized byusing procedures known in the art. Selection inhibits the growth ofnon-modified cells, thus providing an advantage to the growth ofmodified cells, which can be further monitored by tracking the presenceof a fluorescent marker gene or by the appearance of modified explants(modified cells or explants may be green under light in selectionmedium, while surrounding non-modified cells are weakly pigmented). Inplants that develop through shoot organogenesis, the modified cells canform shoots directly, or alternatively, can be isolated and expanded forregeneration of multiple shoots transgenic for the Sugarcanemini-chromosome. In plants that develop through embryogenesis,additional culturing steps may be necessary to induce the modified cellsto form an embryo and to regenerate in the appropriate media. Sugarcaneis generally regenerated through embryogenesis but can also beregenerated by shoot organogenesis.

Useful selectable marker genes are well known in the art and include,for example, herbicide and antibiotic resistance genes including but notlimited to neomycin phosphotransferase II (conferring resistance tokanamycin, paramomycin and G418), hygromycin phosphotransferase(conferring resistance to hygromycin),5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, conferringresistance to glyphosate), phosphinothricin acetyltransferase(conferring resistance to phosphinothricin/bialophos), MerA (conferringresistance to mercuric ions). Selectable marker genes may be transformedusing standard methods in the art.

The first step in the production of Sugarcane plants containing novelgenes involves delivery of DNA into a suitable plant tissue (describedin the previous section) and selection of the tissue under conditionsthat allow preferential growth of any cells containing the novel genes.Selection is typically achieved with a selectable marker gene present inthe delivered DNA, which may be a gene conferring resistance to anantibiotic, herbicide or other killing agent, or a gene allowingutilization of a carbon source not normally metabolized by plant cells.For selection to be effective, the plant cells or tissue need to begrown on selective medium containing the appropriate concentration ofantibiotic or killing agent, and the cells need to be plated at adefined and constant density. The concentration of selective agent andcell density are generally chosen to cause complete growth inhibition ofwild type plant tissue that does not express the selectable marker gene;but allowing cells containing the introduced DNA to grow and expand intoadchromosomal clones. This critical concentration of selective agenttypically is the lowest concentration at which there is complete growthinhibition of wild type cells, at the cell density used in theexperiments. However, in some cases, sub-killing concentrations of theselective agent may be equally or more effective for the isolation ofplant cells containing mini-chromosome DNA, especially in cases wherethe identification of such cells is assisted by a visible marker gene(e.g., fluorescent protein gene) present on the Sugarcanemini-chromosome. Such sub-killing concentrations of the selective agentmay be administered during part or all of the selection timing.

In some species (e.g., tobacco or tomato), a homogenous clone ofmodified cells can also arise spontaneously when bombarded cells areplaced under the appropriate selection. An exemplary selective agent isthe neomycin phosphotransferase II (nptII) marker gene, which iscommonly used in plant biotechnology and confers resistance to theantibiotics kanamycin, 418 (geneticin) and paramomycin. In otherspecies, or in certain plant tissues or when using particular selectablemarkers, homogeneous clones may not arise spontaneously under selection;in this case the clusters of modified cells can be manipulated tohomogeneity using the visible marker genes present on themini-chromosomes as an indication of which cells contain mini-chromosomeDNA.

Regeneration of Modified Plants from Explants to Mature, Rooted Plants

For Sugarcane, regeneration of a whole plant typically occurs via anembryogenic step that is not necessary for plant species where shootorganogenesis is more efficient. The explant tissue is cultured on anappropriate media for embryogenesis, and the embryo is cultured untilshoots form. The regenerated shoots are cultured in a rooting medium toobtain intact whole plants with a fully developed root system. Theseplants are potted in soil and grown to maturity in a greenhouse.

Generally, regeneration and tissue culture of Sugarcane plant parts andwhole plants is challenging as Sugarcane produces phenolic compoundswhile in culture. The present invention provides for methods ofculturing Sugarcane cells and tissues in media containingpolyvinylpyrrolidone (PVP), as described in Example 4. The PVP acts as asink for the phenolic compounds produced by Sugarcane and enhancescallus growth during selection as well as facilitating callus andplantlet regeneration. Furthermore, generation of Sugarcane callus canbe facilitated by delivering to the plant cells and/or tissuesmini-chromosomes of the invention that contain auxin genes. The presenceof the auxin genes will facilitate callus induction of the transformedtissue. The invention also provides for tissue culture methods whichcycle between the liquid culture media and solid culture media in orderto promote the frequency and the morphogenic competence of theregenerable Sugarcane callus.

For plants that develop through shoot organogenesis, regeneration of awhole plant involves culturing of regenerable explant tissues taken fromsterile organogenic callus tissue, seedlings or mature plants on a shootregeneration medium for shoot organogenesis, and rooting of theregenerated shoots in a rooting medium to obtain intact whole plantswith a fully developed root system. These plants are potted in soil andgrown to maturity in a greenhouse.

Explants are obtained from any tissues of a plant suitable forregeneration. Exemplary tissues include hypocotyls, internodes, roots,cotyledons, petioles, cotyledonary petioles, leaves and peduncles,prepared from sterile seedlings or mature plants.

Explants are wounded (for example with a scalpel or razor blade) andcultured on a shoot regeneration medium (SRM) containing Murashige andSkoog (MS) medium as well as a cytokinin, e.g., 6-benzylaminopurine(BA), and an auxin, e.g., α-naphthaleneacetic acid (NAA), and ananti-ethylone agent, e.g., silver nitrate (AgNO₃). For example, 2 mg/Lof BA, 0.05 mg/L of NAA, and 2 mg/L of AgNO₃ can be added to MS mediumfor shoot organogenesis. The most efficient shoot regeneration isobtained from longitudinal sections of internode explants.

Shoots regenerated via organogenesis are rooted in a MS medium. Plantsare potted and grown in a greenhouse to sexual maturity for seedharvest.

To regenerate a whole Sugarcane plant with a Sugarcane mini-chromosome,explants are pre-incubated for 1 to 7 days (or longer) on the shootregeneration medium prior to bombardment with mini-chromosome (seebelow). Following bombardment, explants are incubated on the same shootregeneration medium for a recovery period up to 7 days (or longer),followed by selection for transformed shoots or clusters on the samemedium but with a selective agent appropriate for a particularselectable marker gene (described herein).

Method of Co-Delivering Growth Inducing Genes to Facilitate Isolation ofModified Plant Cell Clones

Another method used in the generation of Sugarcane cell clonescontaining Sugarcane mini-chromosomes involves the co-delivery of DNAcontaining genes that are capable of activating growth of plant cells,or that promote the formation of a specific organ, embryo or plantstructure that is capable of self-sustaining growth. In one embodiment,the recipient Sugarcane cell receives simultaneously the Sugarcanemini-chromosome and a separate DNA molecule encoding one or more growthpromoting, organogenesis-promoting, embryogenesis-promoting orregeneration-promoting genes. Following DNA delivery, expression of theplant growth regulator genes stimulates the plant cells to divide, or toinitiate differentiation into a specific organ, embryo, or other celltypes or tissues capable of regeneration. Multiple plant growthregulator genes can be combined on the same molecule, or co-bombarded onseparate molecules. Use of these genes can also be combined withapplication of plant growth regulator molecules into the medium used toculture the Sugarcane cells, or of precursors to such molecules that areconverted to functional plant growth regulators by the plant cell'sbiosynthetic machinery, or by the genes delivered into the Sugarcanecell.

The co-bombardment strategy of Sugarcane mini-chromosomes with separateDNA molecules encoding plant growth regulators transiently supplies theplant growth regulator genes for several generations of Sugarcane cellsfollowing DNA delivery. During this time, the Sugarcane mini-chromosomemay be stabilized by virtue of its Sugarcane centromere, but the DNAmolecules encoding plant growth regulator genes, ororganogenesis-promoting, embryogenesis-promoting orregeneration-promoting genes will tend to be lost. The transientexpression of these genes, prior to their loss, may give the cellscontaining Sugarcane mini-chromosome DNA a sufficient growth advantage,or sufficient tendency to develop into plant organs, embryos or aregenerable cell cluster, to outgrow the non-modified cells in theirvicinity, or to form a readily identifiable structure that is not formedby non-modified Sugarcane cells. Loss of the DNA molecule encoding thesegenes will prevent phenotypes from manifesting themselves that may becaused by these genes if present through the remainder of Sugarcaneplant regeneration. In rare cases, the DNA molecules encoding plantgrowth regulator genes will integrate into the Sugarcane genome or intothe Sugarcane mini-chromosome.

Alternatively the genes promoting plant cell growth may be genespromoting shoot formation or embryogenesis or giving rise to anyidentifiable organ, tissue or structure that can be regenerated into aSugarcane plant. In this case, it may be possible to obtain embryos orshoots harboring Sugarcane mini-chromosomes directly after DNA delivery,without the need to induce shoot formation with growth activatorssupplied into the medium, or lowering the growth activator treatmentnecessary to regenerate Sugarcane plants. The advantages of this methodare more rapid regeneration, higher transformation efficiency, lowerbackground growth of non-modified tissue, and lower rates of morphologicabnormalities in the regenerated Sugarcane plants (due to shorter andless intense treatments of the tissue with chemical plant growthactivators added to the growth medium).

Determination of Min-Chromosome Structure and Autonomy in SugarcaneAdchromosomal Plants and Tissues

The structure and autonomy of the Sugarcane mini-chromosome in modifiedSugarcane plants and tissues can be determined by methods including butnot limited to: conventional and pulsed-field Southern blothybridization to genomic DNA from modified tissue subjected or notsubjected to restriction endonuclease digestion, dot blot hybridizationof genomic DNA from modified tissue hybridized with differentmini-chromosome specific sequences, mini-chromosome rescue, exonucleaseactivity, PCR on DNA from modified tissues with probes specific to themini-chromosome, or Fluorescence In Situ Hybridization to nuclei ofmodified cells. Table 3 below summarizes these methods.

TABLE 3 Examples of methods to determine mini-chromosome structure andautonomy Assay Assay details Potential outcome Interpretation SouthernRestriction digest of Native sizes and patter

Autonomous or blot genomic DNA* of bands integrated via CEN compared topurified fragment mini-C Altered sizes or pattern Integrated orrearrange

of bands CHEF gel Restriction digest of Native sizes and patter

Autonomous or Southern genomic DNA compar

of bands integrated via CEN blot to purified mini-C fragment Alteredsizes or pattern Integrated or rearrange

of bands Native genomic DNA Mini-C band migrating Autonomous circles or(no digest) ahead of genomic DNA linears present in plant Mini-C bandIntegrated co-migrating with genomic DNA >1 mini-C bands Variouspossibilities observed Exonuclease Exonuclease digestion Signal strengthclose to Autonomous circles assay genomic DNA followe

that w/o exonuclease present by detection of circular No signal orsignal Integrated mini-chromosome by strength lower that w/o PCR, dotblot, or exonuclease restriction digest optional), electrophoresis andsouthern blot (useful fo

circular mini- chromosomes) Mini- Transformation of plan

Colonies isolated only Autonomous circles chromosome genomic DNA into E.from mini-C plants wit

present, native mini-C rescue coli followed by mini-Cs, not fromstructure selection for antibiotic controls; mini-C resistance genes onstructure matches that mini-C of the parental mini-C Colonies isolatedonly Autonomous circles from mini-C plants wit

present, rearranged mini-Cs, not from mini-C structure OR controls;mini-C mini-Cs integrated via structure different from centromerefragment parental mini-C Colonies observed both Various possibilities inmini-C-modified plants and in controls PCR PCR amplification of Allmini-c parts detecte

Complete mini-C various parts of the mi

by PCR sequences present in chromosome plant Subset of mini-c partsPartial mini-C sequenc

detected by PCR present in plant FISH Detection of mini- Mini-Csequences autonomous chromosome sequences detected, free of genom

in mitotic or meiotic Mini-C sequences integrated nuclei by fluorescencedetected, associated wi

in situ hybridization genome Mini-C sequences Both autonomous anddetected, both free and integrated mini-C associated with genom

sequences present No mini-C sequences Mini-C DNA not detected visibl

 by FISH *Genomic DNA refers to total DNA extracted from plantscontaining a mini-chromosome

indicates data missing or illegible when filed

Furthermore, Sugarcane mini-chromosome structure can be examined bycharacterizing mini-chromosomes ‘rescued’ from Sugarcane adchromosomalcells. Circular Sugarcane mini-chromosomes that contain bacterialsequences for their selection and propagation in bacteria can be rescuedfrom a Sugarcane adchromosomal plant or plant cell and re-introducedinto bacteria. If no loss of these sequences has occurred duringreplication of the Sugarcane mini-chromosome in plant cells, themini-chromosome is able to replicate in bacteria and confer antibioticresistance. Total genomic DNA is isolated from the Sugarcaneadchromosomal plant cells by any method for DNA isolation known to thoseskilled in the art, including but not limited to a standardcetyltrimethylammonium bromide (CTAB) based method (Current Protocols inMolecular Biology (1994) John Wiley & Sons, N.Y., 2.3). The purifiedgenomic DNA is introduced into bacteria (e.g., E. coli) using methodsfamiliar to one skilled in the art (for example heat shock orelectroporation). The transformed bacteria are plated on solid mediumcontaining antibiotics to select bacterial clones modified withSugarcane mini-chromosome DNA. Modified bacterial clones are grown up,the plasmid DNA purified (by alkaline lysis for example), and DNAanalyzed by restriction enzyme digestion and gel electrophoresis or bysequencing. Because plant-methylated DNA containing methylcytosineresidues will be degraded by wild-type strains of E. coli, bacterialstrains (e.g. DH10B) deficient in the genes encoding methylationrestriction nucleases (e.g. the mcr and mrr gene loci in E. coli) aresuitable for this type of analysis. Sugarcane mini-chromosome rescue canbe performed on any plant tissue or clone of plant cells comprising amini-chromosome.

Sugarcane Mini-Chromosome Autonomy Demonstration by In SituHybridization (ISH)

To assess whether the Sugarcane mini-chromosome is autonomous from thenative Sugarcane chromosomes, or has integrated into the plant genome,In Situ Hybridization is carried out (Fluorescent In Situ Hybridizationor FISH is particularly well suited to this purpose). In this assay,mitotic or meiotic tissue, such as root tips or meiocytes from theanther, possibly treated with metaphase arrest agents such ascolchicines or nitrous oxide is obtained, and standard FISH methods areused to label both the Sugarcane centromere and sequences specific tothe Sugarcane mini-chromosome. For example, a Sugarcane centromere islabeled using a probe from a sequence that labels all Sugarcanecentromeres, attached to one fluorescent tag (Molecular ProbesAlexafluor 568, for example), and sequences specific to the Sugarcanemini-chromosome are labeled with another fluorescent tag (Alexafluor488, for example). All Sugarcane centromere sequences are detected withthe first tag; only Sugarcane mini-chromosomes are detected with boththe first and second tag. Chromosomes are stained with a DNA-specificdye including but not limited to DAPI, Hoechst 33258, OliGreen, GiemsaYOYO, and TOTO. An autonomous Sugarcane mini-chromosome is visualized asa body that shows hybridization signal with both Sugarcane centromereprobes and Sugarcane mini-chromosome specific probes and is separatefrom the native Sugarcane chromosomes.

Determination of Genes Expression Levels

The expression level of any gene present on the Sugarcanemini-chromosome can be determined by methods including but not limitedto one of the following. The mRNA level of the gene can be determined byNorthern Blot hybridization, Reverse Transcriptase-Polymerase ChainReaction, binding levels of a specific RNA-binding protein, in situhybridization, or dot blot hybridization.

The protein level of the gene product can be determined by Western blothybridization, Enzyme-Linked Immunosorbant Assay (ELISA), fluorescentquantitation of a fluorescent gene product, enzymatic quantitation of anenzymatic gene product, immunohistochemical quantitation, orspectroscopic quantitation of a gene product that absorbs a specificwavelength of light.

Use of Exonuclease to Isolate Circular Sugarcane Mini-Chromosome DNAfrom Sugarcane Genomic DNA

Exonucleases may be used to obtain pure Sugarcane mini-chromosome DNA,suitable for isolation of Sugarcane mini-chromosomes from E. coli orfrom Sugarcane cells. The method assumes a circular structure of theSugarcane mini-chromosome. A DNA preparation containing Sugarcanemini-chromosome DNA and Sugarcane genomic DNA from the source organismis treated with exonuclease, for example lambda exonuclease combinedwith E. coli exonuclease I, or the ATP-dependent exonuclease (QiagenInc). Because the exonuclease is only active on DNA ends, it willspecifically degrade the linear Sugarcane genomic DNA fragments, butwill not affect the circular Sugarcane mini-chromosome DNA. The resultis Sugarcane mini-chromosome DNA in pure form. The resultant Sugarcanemini-chromosome DNA can be detected by a number of methods for DNAdetection known to those skilled in the art, including but not limitedto PCR, dot blot followed by hybridization analysis, and southern blotfollowed by hybridization analysis. Exonuclease treatment followed bydetection of resultant circular Sugarcane mini-chromosomes may be usedas a method to determine Sugarcane mini-chromosome autonomy.

Structural Analysis of Sugarcane Mini-Chromosomes by BAC-End Sequencing

BAC-end sequencing procedures, known to those skilled in the art, can beapplied to characterize Sugarcane mini-chromosome clones for a varietyof purposes, such as structural characterization, determination ofsequence content, and determination of the precise sequence at a uniquesite on the chromosome (for example the specific sequence signaturefound at the junction between a centromere fragment and the vectorsequences). In particular, this method is useful to prove therelationship between a parental Sugarcane mini-chromosome and themini-chromosomes descended from it and isolated from plant cells bymini-chromosome rescue, described above. This method also fostersidentification of specific Sugarcane mini-chromosomes if more than oneunique Sugarcane mini-chromosome is present in a plant cellsimultaneously.

Methods for Scoring Meiotic Sugarcane Mini-Chromosome Inheritance

A variety of methods can be used to assess the efficiency of meioticSugarcane mini-chromosome transmission. In one embodiment of the method,gene expression of genes encoded by the Sugarcane mini-chromosome(marker genes or non-marker genes) can be scored by any method fordetection of gene expression known to those skilled in the art,including but not limited to visible methods (e.g. fluorescence offluorescent protein markers, scoring of visible phenotypes of theplant), scoring resistance of the Sugarcane plants or plant tissues toantibiotics, herbicides or other selective agents, by measuring enzymeactivity of proteins encoded by the mini-chromosome, or measuringnon-visible plant phenotypes, or directly measuring the RNA and proteinproducts of gene expression using microarray, northern blots, in situhybridization, dot blot hybridization, RT-PCR, western blots,immunoprecipitation, Enzyme-Linked Immunosorbant Assay (ELISA),immunofluorescence and radio-immunoassays (RIA). Gene expression can bescored in the post-meiotic stages of microspore, pollen, pollen tube orfemale gametophyte, or the post-zygotic stages such as embryo, seed, orprogeny seedlings and plants. In another embodiment of the method, theSugarcane mini-chromosome can be directly detected or visualized inpost-meiotic, zygotic, embryonal or other cells by a number of methodsfor DNA detection known to those skilled in the art, including but notlimited to fluorescence in situ hybridization, in situ PCR, PCR,southern blot, or by Sugarcane mini-chromosome rescue described above.

FISH Analysis of Sugarcane Mini-Chromosome Copy Number in Meiocytes,Roots or Other Tissues of Modified Sugarcane Plants

The copy number of the Sugarcane mini-chromosome can be assessed in anycell or plant tissue by In Situ Hybridization (Fluorescent In SituHybridization or FISH is particularly well suited to this purpose). Inan exemplary assay, standard FISH methods are used to label theSugarcane centromere, using a probe which labels all Sugarcanechromosomes with one fluorescent tag (Molecular Probes Alexafluor 568,for example), and to label sequences specific to the Sugarcanemini-chromosome with another fluorescent tag (Alexafluor 488, forexample). All Sugarcane centromere sequences are detected with the firsttag; only Sugarcane mini-chromosomes are detected with both the firstand second tag. Nuclei are stained with a DNA-specific dye including butnot limited to DAPI, Hoechst 33258, OliGreen, Giemsa YOYO, and TOTO.Sugarcane mini-chromosome copy number is determined by counting thenumber of fluorescent foci per cell that label with both tags.

Induction of Callus and Roots from Modified Sugarcane Plant Tissues forInheritance Assays

Sugarcane mini-chromosome inheritance is assessed using callus and rootsinduced from transformed Sugarcane plants. To induce roots and callus,tissues such as leaf pieces are prepared from adchromosomal Sugarcaneplants and cultured on a Murashige and Skoog (MS) or Chus's N6 (N6)medium that may contain a cytokinin, e.g., 6-benzylaminopurine (BA), andan auxin, e.g., α-naphthaleneacetic acid (NAA). Any tissue of a modifiedSugarcane plant can be used for callus and root induction, and themedium recipe for tissue culture can be optimized using procedures knownin the art.

Clonal Propagation of Modified Sugarcane Plants

To produce multiple clones of plants from a mini-chromosome-transformedSugarcane plant, any tissue of the plant can be tissue-cultured forshoot organogenesis using regeneration procedures described herein forregeneration of plants from explants. Alternatively, multiple auxiliarybuds can induced from a mini-chromosome-modified Sugarcane plant byexcising the shoot tip, which can be rooted and subsequently be growninto a whole plant; each auxiliary bud can be rooted and produce a wholeplant. Additionally, multiple shoots that result from one plant can besubdivided in culture to produce multiple individual plants.

Scoring of Antibiotic- or Herbicide Resistance in Seedlings and Plants(Progeny of Self- and Out-Crossed Transformants

Progeny seeds harvested from Sugarcane plants comprising amini-chromosome can be scored for antibiotic- or herbicide resistance byseed germination under sterile conditions on a growth media (for exampleMurashigc and Skoog (MS) medium) containing an appropriate selectiveagent for a particular selectable marker gene. Only seeds containing theSugarcane mini-chromosome can germinate on the medium and further growand develop into whole plants. Alternatively, Sugarcane seeds can begerminated in soil, and the germinating seedlings can then be sprayedwith a selective agent appropriate for a selectable marker gene.Sugarcane seedlings that do not contain a Sugarcane mini-chromosome donot survive; only seedlings containing a mini-chromosome can survive anddevelop into mature plants.

Genetic Methods for Analyzing Sugarcane Mini-Chromosome Performance:

Though Sugarcane is typically propagated vegitatively, it is possible touse sexual propagation techniques as well. In addition to directtransformation of a Sugarcane plant with a Sugarcane mini-chromosome,Sugarcane plants containing a Sugarcane mini-chromosome can be preparedby crossing a first Sugarcane plant containing the functional, stable,autonomous Sugarcane mini-chromosome with a second Sugarcane plantlacking the Sugarcane mini-chromosome.

Fertile Sugarcane plants comprising Sugarcane mini-chromosomes can becrossed to other Sugarcane plant lines to study mini-chromosomeperformance and inheritance. In the first embodiment of this method,pollen from an adchromosomal Sugarcane plant can be used to fertilizethe stigma of a non-adchromosomal Sugarcane plant. Sugarcanemini-chromosome presence is scored in the progeny of this cross usingthe methods outlined in the preceding section. In the second embodiment,the reciprocal cross is performed by using pollen from anon-adchromosomal Sugarcane plant to fertilize the flowers of anadchromosomal Sugarcane plant. The rate of Sugarcane mini-chromosomeinheritance in both crosses can be used to establish the frequencies ofmeiotic inheritance in male and female meiosis. In a third embodiment ofthis method, pollen for an adchromosomal plant is used to fertilizeanother or the same adchromosomal plant (e.g. self or siblingpollination). In the fourth embodiment of this method, the progeny ofone of the crosses just described are back-crossed to anon-adchromosomal Sugarcane parental line, and the progeny of thissecond cross are scored for the presence of genetic markers in theplant's natural chromosomes as well as the Sugarcane mini-chromosome.Scoring of a sufficient marker set against a sufficiently large set ofprogeny allows the determination of linkage or co-segregation of theSugarcane mini-chromosome to specific chromosomes or chromosomal loci inthe plant's genome. Genetic crosses performed for testing geneticlinkage can be done with a variety of combinations of parental lines;such variations of the methods described are known to those skilled inthe art.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention andwithout diminishing its intended advantages. It is therefore intendedthat such changes and modifications be covered by the appended claims.

Example 1 Sugarcane Centromere Discovery from Genomic DNA Identificationof Sugarcane Satellite Repeat Sequences

Centromere satellite repeats were amplified from Sugarcane (Saccharumofficinarum×Saccharum spontaneum or Saccharum officinarum) genomic DNAusing standard PCR methods. Briefly, PCR reaction was carried under thefollowing conditions: 1 cycle at 95° C. for 3 minutes, 10 cycles of 94°C. for 15 seconds, 55° C. for 15 seconds, and 72° C. for 30 seconds, and25 cycles at 94° C. for 15 seconds, 52° C. for 15 seconds and 72° C. for30 seconds, followed by 1 cycle at 72° C. for 5 minutes. The sequencesof primers used for amplifying satellite repeats were: forward:5′-gtcacccagcagttccatcgggtgc-3′ (SEQ ID NO: 205 and reverse:5′-actgctgggtgacgtggctcaagt-3′ (SEQ ID NO: 206). After PCR, amplifiedsatellite repeats were cloned into a standard cloning vector (pCR2;Invitrogen Corp.; Carlsbad, Calif.; USA). Colonies with insertions werecultured, DNA was extracted and sequenced. This PCR analysis identified201 satellite sequences, set out at SEQ ID NOS: 1-201.

To determine the consensus of the identified satellite repeat sequences,these sequences were aligned using a DNA sequence alignment program(CONTIGEXPRESS® from VECTOR NTI® (Invitrogen)). The sequences weretrimmed to unit repeat length using the consensus as a template.Sequences trimmed from the ends of the alignment were realigned with theconsensus and further trimmed until all sequences were at or below theconsensus length. The consensus was determined by the frequency of aspecific nucleotide at each position; if the most frequent base is threetimes more frequent than the next most frequent base, it was consideredthe consensus.

The Sugarcane centromere specific retrotransposon sequence CRS(Centromere Retrotransposon in Sugarcane—see Nagaki & Murata, ChromosomeResearch, 2005, 13:195-203) was PCR amplified and sequenced usingprimers located in different region of the CRS sequence. The PCRreaction was carried out as described above using the following primersequences:

(SEQ ID NO: 207) CRSF 5′-gggaagtaca gggacgaaga gc-3′ (SEQ ID NO: 208)CRSF1 5′-actaacaatg cacgggaagg-3′ (SEQ ID NO: 209)CRSF2 5′-gtaggccatg gcagtttgat-3′ (SEQ ID NO: 210)CRSF3 5′-aacacaccac ccaatccaat-3′ (SEQ ID NO: 211)CRSF4 5′-ccaaacaagc gtgttatgat tgt-3′ (SEQ ID NO: 212)CRSF5 5′-aggttatgtg cgtcagtctc ttag-3′ (SEQ ID NO: 213)CRSF6 5′-ggcaaacctg ttgcatactt tag-3′ (SEQ ID NO: 214)CRSF7 5′-accatgtcat aaaactgatg atg-3′ (SEQ ID NO: 215)CRSR 5′-tgcaaccaaa ccaaatcacc ag-3′ (SEQ ID NO: 216)CRSR1 5′-caagcgaaca atctcacgaa-3′ (SEQ ID NO: 217)CRSR2 5′-aaatcatcat cgtgcgcata-3′ (SEQ ID NO: 218)CRSR3 5′-aacacaccac ccaatccaat-3′ (SEQ ID NO: 219)CRSR4 5′-gaacgctcct tgatgacac-3′ (SEQ ID NO: 220)CRSRS 5′-gtacccacta cgcaaatcaa cc-3′ (SEQ ID NO: 221)CRSR6 5′-caacttcagt ttgaccatca gtt-3′

The sequence for CRS is set out as SEQ ID NO: 203. The primer pairs CRSF5′-gggaagtacagggacgaagagc-3′ (SEQ ID NO: 207) and CRSR5′-tgcaaccaaaccaaatcaccag-3′ (SEQ ID NO: 215) can be used to amplify CRSfrom Sugarcane genomic DNA.

BAC Library Construction

A Bacterial Artificial Chromosome (BAC) library was constructed fromSugarcane genomic DNA. The Sugarcane genomic DNA was isolated fromcultivar R570 (P1 504632), a hybrid between S. officinarum and S.spontaneum, and digested with the restriction enzymes Mbo I. Theseenzymes were chosen because they are methylation insensitive andtherefore can be used to enrich BAC libraries for centromere DNAsequences.

Probe Identification and Selection

Three groups of Sugarcane repetitive genomic DNA, including specificcentromere-localized sequences, were initially compiled as candidateprobes for hybridization with the BAC libraries. Four probes were pickedto interrogate the BAC libraries. These probes represent differentgroups of commonly found repetitive sequences in the Sugarcane genome.The four probes were: SCEN, SCRM and High Me/Low Methylation (HiMe andLoMe). The SCEN and SCRM probes were each pooled PCR products. Probeswere prepared and labeled with standard molecular methods. The HiMe andLoMe probes were pooled genomic DNA cut with a methylation-sensitiveenzyme (BfuC1); large DNA fragments were isolated for the “HighMe” probeand small DNA fragments were isolated for the “LowMe” probe. Positivesreported are for the HighMe probe.

Library Interrogation and Data Analysis

The BAC clones from the libraries were spotted onto filters for furtheranalysis. The filters were hybridized with each of the probes toidentify specific BAC clones that contain DNA from the group ofsequences represented by the probes. Hybridization conditions were:hybridization at 65° C. for 12-15 hours and washing three times for15-90 minutes with 0.25×SSC, 0.1% SDS at 65° C. Other exemplarystringent hybridization conditions could be used, such as hybridizationat 65° C. 0.5×SSC 0.25% SDS for 15 minutes, followed by a wash at 65° C.for a half hour.

A total of 18,453 BAC clones from the library was interrogated with eachof the 4 probes (SCEN, SCRM, HiMe, and LoME), and the hybridizationintensities of the BAC clones with each probe were examined to quantifyhybridization intensity for each clone. Scores of 1 to 10 (based on thehybridization intensities, with 10 being the strongest hybridization)were assigned and entered into a spreadsheet for classification. Thespreadsheet contained a total of 3 tables, 1 for each probe used in theinterrogation (values from HiMe and LoMe) probes were entered in asingle table for comparison. Each table contained the hybridizationscores of each BAC clone from the Mbo I library, to one of the 4 probes.Data analysis found BACs that contained different groups of repetitivesequences.

Classification and Selection of BAC Clones for Mini-ChromosomeConstruction

BAC clones containing centromeric/heterochromatic DNA were identified bytheir visual hybridization scores to different probes. The goal was toselect BAC clones that contained a diverse set of various repetitivesequences. Seven classes of centromeric BAC clones were eventuallychosen to cover the broadest possible range ofcentromeric/heterochromatic sequences for Sugarcane mini-chromosome (MC)construction. 658 unique clones that hybridize with one or more of theprobes were isolated from one filter, which comprised 18,432 clones.They fell into classes as set out in Table 4 below.

TABLE 4 Classification of Centromere containing BACs Probe HybridizationRange Class SCEN SCRM HiMe # clones identified I + 472 II + 360 III +398 IV + + 219 V + + 343 VI + + 166 VII + + + 156 * Probes withsignificant hybridization signals as determined by visual scoring areindicated with a “+”

Example 2 Construction of Sugarcane MCs Containing Genomic DNA

A subset of BAC clones identified in Example 1 were grown, and DNA wasextracted for MC construction using a NUCLEOBOND® purification kit fromClontech Laboratories, Inc. (Mountain View, Calif.; USA). To determinethe molecular weight of centromere fragments in the BAC libraries, afrozen sample of bacteria harboring a BAC clone was grown in selectiveliquid media, and the BAC DNA harvested using a standard alkaline lysismethod. The recovered BAC DNA was restriction digested and resolved onan agarose gel. Centromere fragment size was determined by comparing toa molecular weight standard.

Donor DNA Containing Gene Stacks for MC Construction

Several donor DNA plasmids containing gene stacks for testing of MCs inplant tissues were built, varying depending on the specific fluorescentprotein marker used for detection of transgenic events. One set of MCswas built that contained the gene stack from donor plasmid CHROM5798,which genetic elements are set out in Table 5. Another set of MCs wasbuilt that contained the gene stack from donor plasmid CHROM5434, whichgenetic elements are the same as in donor plasmid CHROM5798 except thatthe nuclear localized GFP gene was replaced with an AmCyan (Clontech)fluorescent protein gene. Another set of MCs was built that containedthe gene stack from donor plasmid CHROM5436, which genetic elements arethe same as in donor plasmid CHROM5798, except that the nuclearlocalized GFP gene was replaced with a ZsGreen (Clontech) fluorescentprotein gene.

TABLE 5 Donor Components of CHROM5798 Genetic Size Location Element (bp)(bp) Details YAT1 yeast 2000 6271-8270 PCR amplified YAT1 promoterpromoter from chromosome I of Saccharomyces cerevisiae for expression ofNptII in Sugarcane Arabidopsis 360 5898-6257 PCR amplified ArabidopsisUBQ10 thaliana intron from UBQ10 gene Intron (At4g05320) forstabilization of NptII gene transcript and increase protein expressionlevel NPTII 795 5076-5870 Neomycin phosphotransferase II plantselectable marker Rps16A 489 4524-5012 Amplified from Arabidopsisterminator thaliana 40S ribosomal protein S16 (At2g09990) fortermination of NptII gene Bacterial 817 3525-4341 Bacterial kanamycinselectable kanamycin marker Terminator 6 332 3049-3380 Terminator 6AcGFP (nuc) 831 2084-2914 Nuclear localized green fluores- cent protein.UBQ10 2038  10-2047 PCR amplified Arabidopsis Promoter thaliana promoterfrom UBQ10 gene (At4g05320) for stabilization of DsRedI gene transcriptand increase protein expression level LoxP 34 8290-8319 Recombinationsite for Cre and mediated recombination (Arenski 10802-10831 et al 1983,Abremski et al 1984)

Preparation of Donor DNA for Retrofitting

Cre recombinase-mediated exchange was used to construct Sugarcane MCs bycombining the Sugarcane centromere fragments cloned in pBeloBAC11 withthe donor plasmid CHROM5798 (Table 5). The recipient BAC vector carryingthe Sugarcane centromere fragment contained a loxP recombination site;the donor plasmid contained two such sites, flanking the sequences to beinserted into the recipient BAC.

Sugarcane MCs were constructed using a two-stop method. First, the donorplasmid was linearized to allow free contact between the two loxP sites;in this step the backbone of the donor plasmid is eliminated. In thesecond step, the donor molecules were combined with Sugarcane centromereBACs and were treated with Cre recombinase, generating circularSugarcane MCs with all the components of the donor and recipient DNA.Sugarcane MCs were delivered into E. coli and selected on mediumcontaining kanamycin and chloramphenicol. Only vectors that successfullyere recombined and contained both selectable markers survived in themedium. To determine the molecular weight of the Sugarcane centromerefragments in the Sugarcane MCs, three bacterial colonies from eachtransformation event were independently grown in selective liquid mediaand the Sugarcane MC DNA was harvested using a standard alkaline lysismethod. The recovered Sugarcane MC was restriction digested and resolvedon an agarose gel. Sugarcane centromere fragment size was determined bycomparison to molecular weight standards. When variation in Sugarcanecentromere size was noted, the Sugarcane MC with the largest Sugarcanecentromere insert was used for further experimentation. All 84 MCssubjected to further testing had the features described in Table 6.

TABLE 6 MC constructs tested in sugarcane callus Original Strength ofCEN Donor MC Satellite Strength of Strength of fragment plasmid CHROM#BAC name signal CRS signal FISH signal size (KB) CHROM# 5800 3G17 L Ln/a 90 5798 5801 5B12 H N good 115 5798 5802 18E23 H M good 80 5798 580319H6 M H good 102 5798 5804 24L19 L M good 35 5798 5805 21L3 M M good120 5798 5806 4H1 H N good 90 5798 5807 3O5 M H good 100 5798 5808 1K10L H good 150 5798 5809 17H17 H H good 110 5798 5810 21A4 H H good 1505798 5811 18F12 H H good 110 5798 5812 20H10 H H n/a 150 5798 5813 21B1H H good 130 5798 5814 24J1 H H good 65 5798 5815 19J18 H H good 1005798 5816 17B4 H L good 70 5798 5817 18P24 H L good 120 5798 5818 20A3 HL n/a 160 5798 5819 24F15 H L n/a 120 5798 5820 17C9 H M good 125 57985821 19J8 H N n/a 120 5798 5822 18C14 H N good 130 5798 5823 17P2 H Ngood 110 5798 5824 17M9 H N good 150 5798 5825 24J17 H N n/a 170 57985826 24C6 H N good 75 5798 5827 3I9 L H good 120 5798 5828 22H16 L H n/a135 5798 5829 6C15 L H good 70 5798 5830 20C8 L L good 90 5798 583119K22 L M n/a 105 5798 5832 17E7 L M good 105 5798 5833 24M21 L M n/a130 5798 5834 18J2 M H good 105 5798 5835 17N22 M H n/a 145 5798 58367E2 M H good 230 5798 5837 1L6 M H n/a 90 5798 5838 1P13 M H good 905798 5839 4H14 M L good 70 5798 5840 17E9 M L n/a 110 5798 5841 3P16 M Lgood 100 5798 5842 22I13 M L n/a 130 5798 5843 23F24 M L n/a 90 57985844 1K6 M L good 80 5798 5845 1P14 M L good 135 5798 5846 19H7 M M n/a150 5798 5847 18F16 M M n/a 160 5798 5848 22D19 M M n/a 100 5798 58496C9 M M n/a 120 5798 5850 23I19 M N good 65 5798 5851 3F1 M N n/a 755798 5852 2A7 M N n/a 75 5798 5853 20A22 N N none CEN 100 5798 585419A22 N N n/a 115 5798 5855 17A22 N N n/a 120 5798 5856 21A22 N N noneCEN 80 5798 5857 18A22 N N n/a 120 5798 5858 1A4 L H n/a 65 5798 58591M10 N N n/a 110 5798 5860 7D11 M M n/a 80 5798 5861 7J24 M L n/a 1005798 5862 1P1 H L n/a 125 5798 5863 21B11 H N good 150 5798 5864 8O2 M Lgood 70 5798 5865 8I7 M H good 135 5798 5866 17H17 H H good 110 54345867 21A4 H H good 140 5434 5868 19J18 H H good 95 5434 5869 18P24 H Lgood 120 5434 5870 17C9 H M good 120 5434 5871 18E23 H M good 85 54345874 4H14 M L good 85 5434 5876 17P2 H N good 100 5434 5878 6C15 L Hgood 80 5434 5881 17C9 H M good 120 5436 5882 17P2 H N good 100 54365883 4H14 M L good 85 5436 5884 18E23 H M good 85 5436 5885 6C15 L Hgood 80 5436 5886 18P24 H L good 120 5436 5887 19J18 H H good 95 54365888 21A4 H H good 140 5436 5889 17H17 H H good 110 5436 H indicateshigh hybridization signal in the original filter hybridization, Mindicates medium signal, and L indicates low signal. N indicates nosignal was observed. In column labeled “Strength of FISH signal,” “good”indicates strong hybridization to centromeres observed in root tipspread, n/a indicates “not determined,” and “none CEN” means nohybridization was observed to the centromeric region of any chromosomes.

Example 3 MC Delivery into Sugarcane Cells and Regeneration

The Sugarcane MCs from Example 2 were tested in several Sugarcane cells,including Saccharum officinarum and a hybrid between S. officinarum andS. spontaneum, and the procedure was optimized for antibiotic selection,cell pre-treatments, and bombardment conditions. MCs were tested both inleaf-roll tissue directly, or callus tissue that was initiated fromleaf-rolls. The presence of MCs was determined both by direct molecularassays or indirect measurement of fluorescent cells. Preliminary resultsidentified several MCs that successfully generated fluorescent cellclusters in Saccharum cells.

Sugarcane Transformation, Selection and Regeneration.

Prior to delivery of the two-gene stack containing MCs from Example 2,Sugarcane callus was initiated from leaf roll tissue. Sugarcane topswere collected from greenhouse-grown plants for preparing explants. TheSugarcane tops (minimally 3-6 months old) were cut below the highestvisible node. The older leaves (approx 3-4) were removed until theinternode was visible and cut about 2″ below this internode, anddisinfected by submerging in 20% bleach for 20 min (5-10 tops in 3 Lbleach solution). Subsequently, the cane tops were rinsed with steriledistilled water 3-4 times to remove excess bleach. In a tissue culturehood, more external leaves were removed from the cane tops, and the topportion was cut off leaving about 10 to 12 cm above the internode and 1cm below the internode. Thin stem sections (approx 1 to 1.5 mmthickness) were sliced from the lower edge of the internodes. Duringthis process, tools were frequently dipped in antioxidant mixture(PhytoTechnology Laboratories; Shawnee Mission, Kans.; USA) to avoidbrowning at cut sites. Those sections with orange centers were avoided,and only those sections with green centers were used for callusinduction. Approximately 9 pieces of thin stem sections were placed perplate on MS3 Medium (Murashige and Skoog, 1962. Physiologia Plantarum,15:472-497), supplemented with 500 mg/L casein hydrolysate, 20 g/Lsucrose and 3 mg/L 2,4-D, pH to 5.8 and solidified with 2.5 g/LGELRITE@(Sigma-Aldrich; Saint Louis, Mo.; USA) or 6 g/L Phytoblend(Caisson Laboratories; North Logan, Utah; USA). The callus wassub-cultured once after a 15-day interval onto the same medium or MS1(MS; 4.3 g/l MS salts and vitamins supplemented with 20 g/l sucrose, 0.5g/l casein, 1 mg/l 2,4-D, pH to 5.8) medium. Callus was generallysufficiently established for bombardment after 2-3 months. Prior tobombardment with MCs, the white, nodular and embryogenic calli weresubcultured for 3-4 days on MS3 Medium, and then transferred ontoSugarcane Osmotic Medium (SCOM) prior to bombardment (4-5 hours at 28°C.). Sugarcane Osmotic Medium consists of MS3 medium supplemented with500 mg/L casein hydrolysate, 20 g/L sucrose and 3 mg/L 2,4-D. pH to 5.8and solidify with 2.5 g/L GELRITE® or 6 g/L Phytoblend with the additionof 36.4 g/L sorbitol, and 36.4 g/L mannitol.

Precipitation of MC DNA onto gold particles for the purpose of deliveryusing the biolistic method was performed as follows: 1.8 mg of sterile,washed gold (0.6 μM diameter was preferred) was combined with desiredamount of MC DNA (in 1×TE). Careful handling of DNA was critical; widebore tips were used for all pipetting, and solutions were preferentiallydispensed into the bottom of the tube to assist with the gentle mixingprocess. The volume was brought to 250 μl with cold (4° C.) sterilewater and 250 μl of cold (4° C.) 2.5M CaCl₂ was added immediately,followed by addition of 50 μl of filter sterilized 0.1M Spermidine (freebase, filter sterilized). The mixture was gently finger vortexed 1-2× toensure even mixing of all solutions, and DNA was allowed to precipitateonto the gold particles on ice for 1.5 hours; with finger vortexing 1-2×after 45 min. The gold/DNA mixture was pelleted (5 min, 800 rpm, RT) andwashed once with 100% ethanol, and 36 μl 100% ethanol was added to thegold/DNA pellet, and mixed gently. Typically 6 μl of gold/DNA/ethanolwas used per macrocarrier (i.e., one bombardment shot). The absolutenumber of molecules delivered per shot was varied by precipitating avarying amount of DNA onto the gold particles.

Bombardment conditions using the BioRad PDS-1000/He biolistictransformation system Bio-Rad Laboratories; Hercules, Calif.) were asfollows. A rupture disk rating of 900-1800 psi; 1100 or 1300 psi waspreferred, with one shot per plate. The preferred gap distance (distancefrom rupture disk to macrocarrier) was 6 mm. The target shelf for tissuewas L2-L4; L2 (3rd shelf from the bottom) was preferred. Vacuum pressureof 27.5-28 in Hg; 27.5 in Hg was preferred. These bombardment conditionswere tested with R570 callus, other conditions (pressure, rupture diskrating, gap distance, target shelf, duration of osmotic and resttreatments, etc.) can of course be modified for other genotypes and/ortissues. Following bombardment, callus was allowed to recover at 28° C.in the dark for an additional 12-18 hours on SCOM. Tissues weretransferred onto MS3 medium for recovery for 3-4 days, and the number ofcalli present on each plate used for bombardment was counted. At the endof the recovery period, calli were checked for transient expression offluorescent marker genes under the microscope, and the transienttransformation efficiency was calculated.

Transient expression of MC encoded green fluorescent protein (GFP) geneAcGFPnuc was demonstrated in Sugarcane (cv. L97-128) callus induced fromimmature leaf tissue. Approximately 2.5×10⁹ DNA molecules for 9different MC DNAs were delivered into the callus tissues 4 hours afterosmotic treatment per plate, and the bombarded tissues were examined forGPF expression 4 days later. The results are summarized in Table 7.Calli expressing GFP were observed in 6 out of the 9 MCs delivered inthis experiment, with a frequency ranging from 1.7% for MC CHROM5889 to20% for MC CHROM5886. However, no GFP expressing calli were observed for3 MC constructs. These results demonstrated that MC DNA had beensuccessfully delivered into sugarcane callus cells and the MC encodedfluorescent protein gone AcGFPnuc was expressed and biologicallyfunctional.

TABLE 7 Transient expression of AcGFPbuc in Sugarcane MC containingtransgenic callus # of calli % GFP BAC # of plates # of calli expressingexpressing MC # Cen observed observed GFP callus 5881 17C9 7 140 5 3.65882 17P2 9 92 4 4.3 5883 4H14 12 136 0 0.0 5884 18E24 12 140 6 4.3 58856C15 10 120 3 2.5 5886 18P24 4 60 12 20.0 5887 19J18 5 82 0 0.0 599821A4 5 94 0 0.0 5889 17H17 6 120 2 1.7

For selection of transgenic events, bombarded calli were transferredonto sub-lethal selection medium (ChromMS3G30), and culture at 28° C.,in the dark, for 2 weeks. ChromMS3G30 medium consists of MS3 mediumsupplemented with 30 mg/l G418 sulfate (Geneticin (Sigma)) afterautoclaving. The calli were broken up into small pieces and transferredonto lethal selection medium (second round of selection) MS3G50, andculture at 28° C., in the dark, for 4 weeks. MS3G50 Medium consists ofMS3 medium supplemented with 50 mg/l G418 sulfate after autoclaving.Tissue growth was visually assessed to identify resistant callus.Resistant calli were subcultured for another round of selection onChromMS3G50 for an additional 4 weeks.

For plant regeneration, surviving calli (putative resistant calli) weretransferred onto RSCG25 medium to initiate regeneration and werecultured at 26° C., low light (16 hour day length, 26° C.) for 3-4weeks. RSCG25 Medium consists of MS3 medium supplemented with 500 mg/Lcasein hydrolysate, 20 g/L sucrose and 0.5 mg/L kinetin, pH to 5.8 andsolidified with 2.5 g/L GELRITE@, and further supplemented with 25 mg/LG418 sulfate after autoclaving. Developing plantlets were transferred toRtSC medium in sundae cups (Solo Cup Company; Lake Forest, Ill.; USA)for plantlet growth and root development and cultured at 26° C., 16 hourday length. RtSC medium consists of MS medium supplemented with 25 g/Lsucrose, pH to 5.8 and solidify with 2.5 g/L GELRITE®, furthersupplemented with 20 mg/L G418 sulfate after autoclaving. Finally,plantlets were transferred into pre-moistened soil-less mix (LCI, BFGSupply Company; Joliet, Ill.; USA) under a humidome in an 18-well flatin a growth chamber (28° C., 16 hour day length). The dome was crackedopen slightly to slowly reduce humidity 3-4 days after transplanting.The dome was removed completely 2 days later and plantlets weretransferred to a greenhouse (28° C., 16 hour day length). Plants werewatered from trays beneath the pots when the soil began to dry.

Identification of MC Containing Transgenic Events

More than 1200 putative transgenic Sugarcane events were generated frommini-chromosome transformation. A total of 920 (˜76%) of putativetransgenic calli events were analyzed using diagnostic PCR for theseveral DNA fragments carried on the gene stack. The presence or absenceof the nptII, AcGFPnuc or ZsGreen (depending on the mini-chromosome usedin transformation), and the ubiquitin (UBQ10) promoter was determinedand compared to amplification of the endogenous genomic internal controlADH. The events thus screened cover a collection of 51 MCs with anaverage size ranging between 65 and 187 kb. Transgenic events werederived from the bombardment of six different sugarcane genotypes (R570,L97-128, Q117, NCo310, Pindar and Q63). Of the 920 events, 33% werederived from the callus bombarded with the DNA concentration of 1×10⁹molecules (90-200 ng of DNA) per shot, 8.7% from 2.5×10⁹ molecules(200-500 ng of DNA), 37.6% from 5×10⁹ molecules (450 ng-1 μg of DNA) and6.2% from 1×10° molecules (800 ng-1.2 μg of DNA) per shot. The putativetransgenic events were obtained after selecting the bombarded calli ondifferent levels of G418 concentration ranging from 22.5 mg to 50 mg/l(100% potency) for a period of 4-5 months with a minimum of 4 rounds ofselection. For PCR screening, total genomic DNA was isolated fromapproximately 40-60 mg of callus tissue by the DNA preparation method ofKrysan et al. (Krysan P I I, Young J C, Tax F, Sussman M R (1996)Identification of transferred DNA insertions within Arabidopsis genesinvolved in signal transduction and ion transport. Proc Natl Acad SciUSA 93: 8145-8150). The concentration of DNA was measured, normalizeduniformly and used for either single-step or multiplex PCR analysis. Theoptimized PCR conditions, primers and reagents that were used fordetecting the 4 PCR fragments in all the calli events generated is asfollows.

Single-step PCR conditions used an initial denaturation at 95° C. for 2minutes, followed by 35 cycles each consisting of 0.3 minutesdenaturation at 94° C., 0.3 minutes annealing at 52° C., and 1.2 minutesextension at 72° C., followed by a final extension for 4 minutes at 72°C., after which the samples were kept at 4° C. indefinitely. The PCRreaction was performed in a total volume of 25 μl, consisting of 19.2 μlwater, 2.5 μl 10×NEB Buffer, 0.4 μl dNTP mix (40 Mm), 0.2 μl F-Primer(20 μM), 0.2 μl R-primer (20 μM), 0.50.2 μl NEB Taq Polymerase and 2 μlDNA (100-200 ng). Multiplex PCR conditions used an initial denaturationat 95° C. for 1 minutes, followed by 40 cycles each consisting of 0.1minutes denaturation at 95° C., 0.1 minutes annealing at 55° C. (andincreasing 0.1° C. with each subsequent cycle), and 1.5 minutesextension at 72° C., followed by a final extension for 5 minutes at 72°C., after which the samples were kept at 4° C. indefinitely. The PCRreaction was performed in a total volume of 25 μl, consisting of 15 μlwater, 5 μl 5× Multiplex Master Mix (New England Biolabs; Ipswich,Mass.; USA), 2 μl F-Primer (20 μM), 2 μl R-primer (20 μM), and 1 μl DNA(100-200 ng). The expected PCR product sizes were 470 bp (ADH), 662 bp(AcGFPnuc), 886 bp (UBQ10), 1000 bp (nptII), and 924 hp (ZsCreen). ThePCR reaction was carried out as described above using the followingprimer sequences:

(SEQ ID NO: 242) ADH1 CHSL-285 5′-aagtcggcag agagcaacat-3′(SEQ ID NO: 243) ADH1 CHSL-286 5′-cagatgcaaa cccaacacac-3′(SEQ ID NO: 244) AcGFPnuc CHSL-132 5′-cgattttctg ggtttgatcgtt ag-3′(SEQ ID NO: 245) AcGFPnuc CHSL-199 5′-cattgtgggc gttgtagttg-3′(SEQ ID NO: 246) UBQ10 CHSL-468 5′-gttgtggtty gtgctttcct-3′(SEQ ID NO: 247) UBQ10 CHSL-469 5′-ccactttgac gccgtttatt-3′(SEQ ID NO: 248) NPTII CHSL-132 5′-cgattttctg ggtttgatcgttag-3′(SEQ ID NO: 249) NPTII CHND7 5′-gaactcgtca agaaggcgata-3′(SEQ ID NO: 250) ZsGreen CHSL-132 5′-cgattttctg ggtttgatcgttag-3′(SEQ ID NO: 251) ZsGreen CHSL-201 5′-tcagggcaat gcagatcc-3′

Among the 920 events analyzed, 358 events (38.9%) showed the presence ofall three PCR amplicons (nptII, AcGFPnuc or ZsGreen, and UBQ10)(summarized in Table 8). Based on this analysis the escape frequency isaround 61%. The observed escape rate appears the highest in genotypeR570, in which most of the transformations were performed.

TABLE 8 Sugarcane MC containing transgenic callus Events Positive eventsMC # analyzed for all amplicons 5800 24 12 5801 9 0 5802 42 18 5803 5 15804 10 2 5805 3 1 5806 4 1 5809 11 3 5810 7 5 5812 4 0 5814 88 62 581629 14 5817 42 12 5819 75 43 5820 53 42 5821 4 1 5822 18 1 5823 4 0 582412 0 5825 7 0 5827 6 0 5830 5 0 5834 20 3 5835 4 1 5837 12 1 5839 7 05840 3 0 5842 2 0 5844 7 0 5846 14 0 5850 14 11 5851 14 0 5852 5 0 585426 3 5856 11 3 5857 6 1 5858 25 1 5859 14 0 5860 26 4 5862 11 0 5863 110 5864 50 8 5873 2 2 5874 14 8 5881 47 44 5882 19 13 5883 12 4 5884 3516 5885 23 8 5888 2 2 5889 22 7 Total 920 358

MC transgenic events were obtained in all genotypes tested, andsignificant genotype dependence was noted for both transformationefficiency and escape rate for G418 selection. Table 9 summarizestransformation results for several genotypes.

TABLE 9 Sugarcane MC transgenic events in multiple genotypes No. ofputative No. of PCR Escape frequency Genotype events produced positiveevents (%) R570 554 94 83 Q117 151 95 37 L97-128 182 142 22 NCo310 6 6 0Pindar 23 18 21.7 Q63 4 3 25 Total 920 358

Evaluation of Autonomous MCs

To evaluate whether the candidate Sugarcane MCs were maintainedautonomously, fluorescence in situ hybridization (FISH) was performed onmitotic metaphase chromosome spreads from callus tissue. FISH wasperformed essentially as described in Kato et al. Proc. Natl. Acad. Sci.U.S.A. 101: 13554-13559, 2004, using probes labeled with ALEXA FLUOR®488 (“Alexa488”) and ALEXA FLUOR®568 (“Alexa568;” Invitrogen). Alexa488labeled CHROM5798 DNA was used as a MC-specific probe. Alexa568 labeledpBeloBAC11 DNA was used as a second MC-specific probe. Alexa568 labeledPCR amplified centromere sequences from BAC 18E23 were used as acentromere-specific probe. The latter probe was also expected to staincentromere regions on the endogenous chromosomes.

For FISH evaluation, freshly growing callus tissue was collectedfollowing a recent transfer to fresh media. Depending on genotype,different morphologies were apparent. Generally, tissue was nodular andfirm, and met forceps with resistance. Using forceps or scalpel, a verysmall cluster of nodules was excised and transferred to a 1.7 mLmicrofuge tube. A few microliters of dH₂O were added to the tube to keepthe tissue moist. The tube was covered with cap containing a smallpuncture to allow exposure to nitrous oxide in next step. Callus tissuewas placed in the pressure chamber under 160 psi for 4.5 hours. Tissuewas fixed in 90% acetic acid, and spread onto poly-lysine coated glassslides by squashing thin cross sections. Following hybridization, slideswere counter-stained with DAPI (0.04 mg/ml) and ≧15 metaphase cells wereevaluated per callus using a Zeiss Axio-Imager (Carl Zeiss MicroImaging,Inc.; Thornwood, N.Y.; USA) equipped with rhodamine, FITC, and DAPIfilter sets (excitation BP 550/24, emission BP 605/70; excitation BP470/40, emission: BP525/50; and excitation G 365, emission BP 445/50,respectively). Gray-scale images were captured in each panel, merged andadjusted with pseudo-color using Zeiss AxioVision (Version 4.5; CarlZeiss MicroImaging, Inc.) software; fluorescent signals fromdoubly-labeled MCs were detected in both the red and green channels.

Extra-chromosomal signals were considered to indicate autonomousSugarcane MCs if the images showed co-localization of the Alexa488(green) and Alexa568 (red) signals within 1 nuclear diameter of theendogenous metaphase Sugarcane chromosomes, and the signals were clearlydistinct from the DAPI-stained host chromosomes. Typical Autonomous MCsignals in FISH hybridization show overlapping distinct Alexa488 (green)and Alexa568 (red) signals that in computer-generated merged images, theoverlap shows a yellow signal. Integrated constructs result in twodistinct FISH signals, each on a replicated metaphase chromatid, andusually these FISH signals do not overlap with the centromere region.Autonomous MCs were found to co-exist in the presence of integratedconstructs, indicating the ability of a specific MC to produce a purelyautonomous event in transgenic lines obtained in parallel with the eventcharacterized here, or obtained from future transformation experimentsunder different transformation conditions. Table 10 summarizes thepreliminary FISH evaluation of selected transgenic lines that wereconfirmed to contain MCs identified by PCR, and demonstrated that bothautonomous only (category A) and autonomous and integrated events(category A+I) were obtained for a significant number of Sugarcane MCs.

TABLE 10 Preliminary FISH evaluation of Sugarcane MC transgenic eventsAutonomous Integrated CHROM# Event ID# copy copy Category 5802RC5802-85-12-5 + − A 5802 RC5802-85-14-2 + − A 5802 RC5802-85-14-2 + − A5802 RC5802-85-16-1 + − A 5814 BC5814-109-6-1 + − A 5814BC5814-109-9-1 + − A 5819 AC5819-109-29-1 + − A 5819 AC5819-109-30-2 + −A 5820 BC5820-105-13-1 + − A 5820 BC5820-105-13-1 + − A 5820RC5820-74-6-3 + − A 5824 RC5824-77-29-1 R + − A 5837 RC5837-88-13-1 + −A 5840 RC5840-99-10-1 + − A 5860 RC5860-75-13-1 + − A 5802RC5802-85-14-5 + + A + I 5809 RC5809-79-4-1 + + A + I 5810BC5810-112-23-1 + + A + I 5810 BC5810-112-23-2 + + A + I 5814AC5814-109-12-2 + + A + I 5814 AC5814-109-14-1 + + A + I 5814AC5814-109-14-8 + + A + I 5814 AC5814-109-15-10 + + A + I 5814AC5814-109-15-5 + + A + I 5814 RC5814-101-16-1 + + A + I 5816BC5816-109-21-5 + + A + 1 5816 BC5816-109-21-6 + + A + I 5817PC5817-104-1-1 + + A + I 5817 PC5817-104-2-1 + + A + I 5817PC5817-104-5-1 + + A + I 5819 AC5819-109-26-1 + + A + I 5819AC5819-109-27-1 + + A + I 5819 AC5819-109-29-1 + + A + I 5819AC5819-109-29-4 + + A + I 5819 BC5819-105-3-023 + + A + I 5820BC5820-105-11-1 + + A + I 5820 BC5820-105-15-1 + + A + I 5820BC5820-105-15-1 + + A + I 5820 BC5820-105-15-1 + + A + I 5834RC5834-80-2-1 + + A + I 5850 RC5850-74-16-2 + + A + I 5850RC5850-74-16-2 + + A + I 5850 RC5850-74-16-2 + + A + I 5850RC5850-74-18-8 + + A + I Events with high quality FISH signals asdetermined by visual scoring are indicated with a “+”, a clear absenceof signal is indicated by “−”.

Example 4 Construction of Sugarcane MCs Containing Synthetic Arrays ofRepeat Sequence

A synthetic array of the satellite repeat sequences was generated usingPCR and directional cloning. A block of 4 Sugarcane satellite repeatswere PCR amplified and sequenced. The sequence is set out as SEQ ID NO:204. This sequence was used as the basis for building the syntheticarray. Several arrays were constructed ranging in size between 8 kb and25 kb. Two Sugarcane MCs containing specific synthetic arrays of 18 kb(CHROM5613) and 25 Kb (CHROM5616) were used in further testing.

The Sugarcane MCs also contained either 5 or 8 stacked exogenous genes.The 5- or 8-gene stacks were based on the 2-gene stack construct fromdonor plasmid CHROM5738. The genetic elements within the donor plasmidCHROM5738 are the same as in donor plasmid CHROM5798 except that thenuclear localized GFP gene was replaced with a nuclear localized DsRed2fluorescent protein gene (DsRed2+NLS), described in Table 11.

TABLE 11 Donor Components of CHROM5738 Size Genetic (base LocationElement pair) (bp) Details YAT1 yeast 2000 7110-9109 PCR amplified YAT1Promoter promoter from chromo- some I of Saccharomyces cerevisiae forexpression of NptII in Sugarcane Arabidopsis 360 9123-9482 PCR amplifiedArabidopsis UBQ10 Intron thaliana intron from UBQ10 gene (At4g05320) forstabilization of NptII gene transcript and increase protein expressionlevel nptII 795  9510-10304 Neomycin phosphotransfer- ase II plantselectable marker Rps16A 489 10368-10856 Amplified from Arabidopsisterminator thaliana 40S ribosomal protein S16 (At2g09990) fortermination of NptII gene Bacterial 817 11039-11855 Bacterial kanamycinKanamycin selectable marker Terminator 6 332 12000-12331 Terminator 6DsRed2 + NLS 780 12466-13245 Nuclear localized red fluorescent proteinfrom Discosoma sp. (Matz, M et. al Nat Biotechnol 1999 December; 17(12):1227). UBQ10 2038 13282-15319 PCR amplified Arabidopsis Promoterthaliana promoter from UBQ10 gene (At4g05320) for stabilization ofDsRedI gene transcript and increase protein expression level LoxP 347057-7090 Recombination site for Cre and mediated recombination15335-15368 (Arenski et. al 1983, Abremski et. al 1984)

The 5-gene stack included the marker genes Anthocyanin, ZsGreen, andZsYellow in addition to NptII, DsRed in CHROM5738. The 8-gene stackincluded those genes in the 5-gene stack plus three additional genesfrom the Agrobacterium tumefaciens tumor-inducing (Ti) pathway. Thesewere the iaaM (Trp mono-oxygenase), iaaH (Indole-3-acetamide hydrolase),and ipt (AMP iso-pentenyl transferase) genes.

In order to investigate whether the Sugarcane MCs could accommodate alarge number of genes, Sugarcane MCs containing a gene stack, asynthetic array of repeat nucleotide sequence and about 20 kb of A.thaliana DNA was constructed. The total size of these Sugarcane MCsranged between 82 kb and 87 kb. In addition, Sugarcane MCs with a genestack with 2 genes in addition to a 28 kb synthetic Sugarcane centromererepeat array and an approximately 50 kb insertion of A. thaliana DNA wasconstructed using the methods described above. The Sugarcane MCdemonstrated that the Sugarcane MCs of the invention can accommodate alarge payload of genes, as 50 kb of the A. thaliana DNA includes a widevariety of genes. The total size of these MCs were 82 and 87 kb.

Sugarcane Transformation and Regeneration with Synthetic MCs.

To enhance the efficiency with which Sugarcane cells transformed withSugarcane MCs can be regenerated into Sugarcane plants, Sugarcane MCscontaining the auxin gone pathway were delivered into fullydifferentiated leaf rolls rather than undifferentiated tissue, e.g.,embryos. In addition, the growth conditions were altered in order tofacilitate generation of transformed Sugarcane callus. FunctionalTesting of Sugarcane MCs Using Transient Assays

Sugarcane (variety R570; Saccharum oficinarum×Saccharum spontaneum) wasgrown in the greenhouse for 6 months without floral initiation due tothe growth time as well as the day-length settings on greenhousesupplemental lighting. Stalks from several (clonal) plants were used togenerate leaf rolls that were purely leaf tissue and did not include anydeveloping meristematic tissue.

Sugarcane MCs with a synthetic Sugarcane centromere were delivered toleaf rolls. One Sugarcane MC contained an 8-gene stack (denoted hereinas the “8-gene MC”), and one Sugarcane MC contained a 5-gene stack(denoted herein as the “5-gene MC”). These Sugarcane MCs are describedin detail in Example 3. In addition, a control plasmid (lacking acentromere) containing eight genes was also delivered, in which the8-gene stack is identical to that delivered on the 8-gene Sugarcane MC.

The 8-gene Sugarcane MC included A. tumefaciens tumor inducing (Ti)pathway genes (iaaM, iaaH, and ipt). The inclusion of these genesminimized the time the transformed cells were in tissue culture. IaaMconverts Trp into indole-3-acetamide, which IaaH converts into auxin.Isopentenyl transferase (Ipt) converts 3′,5′-adenosine monophosphate(AMP) into a cytokinin. The presence of the hormone auxin was used incell culture to stimulate plant cells to form callus. Media with auxinpromotes callus growth from plant cells whereas plant cells cultured onmedia lacking auxin either germinate (for embryogenic material) or areunable to grow (non-embryogenic or meristematic tissue such as leaftissue). Thus, the 8-gene MC induced callus formation withoutsupplementing the media with auxin.

A biolistic delivery method using dry gold particles was carried out todeliver MCs to the Sugarcane leaf rolls. For this method, Sugarcane MCDNA (in 1×TE) was precipitated onto 2.1 mg of sterilized and washed 0.6pt gold particles. The DNA-containing gold particles were re-suspendedin cold sterile water and 2.5 M CaCl₂. Filter-sterilized 0.1 Mspermidine (free base) was added to the mixture. Subsequently, themixture was allowed to precipitate on ice for 1.5 hours, with gentlefinger vortexing (3×) after 45 minutes. The precipitated DNA was thenwashed with 100% ethanol, resuspended in 100% ethanol which was allowedto fully evaporate prior to bombardment.

The apical region of the Sugarcane stem was collected (20-30 cm long),after removing the outermost mature leaves, the remaining leaves weresterilized by submersion in a solution of 50 ml bleach in 1 liter ofwater for 10 minutes. The remaining mature leaves were asepticallyremoved and the young inner immature leaves were sliced intosections/discs approximately 2-3 mm thick. The leaf rolls were placed inSCOM at 28° C. for 4-5 hours before bombardment.

The 3 constructs wore each initially tested by delivery into 16 leafrolls. For delivery, the leaf rolls were bombarded with the MC DNA usingthe BioRad PDS-1000/He with a rupture disk rating of 900-1800 psi (1350psi was preferred with one shot per plate). The gap distance (distancefrom rupture disk to macrocarrier) was 6 mm. Target shelf for tissue wasL2-L4; L2 or L3 was preferred. The vacuum pressure of 25-29 in Hg; 27.5in Hg was preferred. The bombarded leaf rolls were stored at 28° C.(dark) for an additional 16-18 hours on SCOM.

Subsequently, the bombarded leaf rolls were transferred to MSO (4.3 g/lMurashige and Skoog (MS) salts and vitamins, supplemented with 20 g/lsucrose, 0.5 g/1 casein, with NO 2,4-D, pH to 5.8 and solidified with 2g/L GELRITE®) and stored at 28° C. in the dark for 2 weeks. The leafrolls were visually assessed for callus production 2 and 4 weeks afterbombardment. Control leaf rolls not subjected to bombardment, and theleaf rolls that were bombarded with the 5-gene stack MC showed no signof growth or callus formation. Two of the 16 bombarded leaf rolls thatwere bombarded with the 8-gene stack MC produced callus (12.5% of thetotal explants).

Callus from the tissue was phenotypically evaluated for DsRed expressionusing a fluorescent dissecting microscope. DsRed was observed in thetissue. The resulting calluses were transferred to RegenerationSugarcane Medium (RSCM; 43 g/l MS salts and vitamins supplemented with20 g/l sucrose, 0.5 g/l casein, 0.5 mg/l kinetin. pH to 5.8 and solidifywith 2 g/L GELRITE®) in low light (16 hour day length, 26° C.) toinitiate regeneration. This media did not contain auxin. After 2additional weeks of culture, callus had grown, but had also started todifferentiate into root (primarily) and shoot material. Suchdifferentiation is not expected in the presence of auxin, suggestingeither silencing or loss of the 3 genes from the A. tumefaciens Tipathway. After 2 additional weeks on media, PCR evaluation of thismaterial was done for presence of the DsRed gene. PCR results werenegative, which further suggested loss of the entire MC and verifiedthat the genes were not silenced. Because of the loss of the entire MC,these tissues were not regenerated into plants.

The advantages of including the genes of the Ti pathway on a MC are thatthe non-meristematic tissues were transformed and the need for callusinitiation prior to DNA delivery was eliminated. In addition, the timein culture was reduced and as a result somaclonal variation, endogenouschromosome number changes and the like were also reduced. Furthermore,the inclusion of the Ti pathway genes eliminated the need for selectablemarker genes. There was an observed 12.5% transformation efficiency ofSugarcane in the initial experiments.

In a separate experiment, 5-gene (NptII, DsRed, Anthocyanin, ZsGreen,and ZsYellow) MCs were delivered into Sugarcane callus generated fromSugarcane variety R570; (Saccharum officinarum×Saccharum sponlaneum)These MCs were delivered to the Sugarcane callus cells using the wetbiolistic method as described above. In brief, the droplet containingthe MC DNA-coated particles was delivered to cells using a RioRadPDS-1000/He with a rupture disk rating of 400-1800 psi (650 psi waspreferred with one shot per plate) adapted for a filter holder, whichwas attached to a base which was itself attached to a rupture diskholder used to hold the rupture disk to the helium egress tube forbombardment. The gas discharge directly displaced the DNA/gold dropletfrom the filter holder and accelerated the particles and their DNA cargointo the tissue being bombarded.

Following delivery, callus from the tissue was phenotypically evaluatedfor DsRed expression using a fluorescent dissecting microscope. DsRedwas observed in the tissue. The resulting calluses were transferred toSelection Sugarcane Medium MS3-50; (4.3 g/l MS salts and vitaminssupplemented with 20 g/l sucrose, 0.5 g/l casein, 3.0 mg/l 2,4-D, 0.5g/l polyvinylpyrrolidone (PVP). pH to 5.8 with 2 g/l GELRITE®; furthersupplemented with 50 mg/l G418 after autoclaving) for initial selectionfor 2 weeks. All calluses were subsequently transferred to additionalselection on Selection Sugarcane Medium MS3-75 (4.3 g/l MS salts andvitamins supplemented with 20 g/l sucrose, 0.5 g/l casein, 3.0 mg/l2,4-D, 0.5 g/l polyvinylpyrrolidone (PVP) pH to 5.8 with 2 g/l GELRITE®;further supplemented with 75 mg/l G418 after autoclaving) for 4additional weeks. Tissue was then visually assessed for Sugarcane callustissue that was able to grow on this selection. Those identified eventswere transferred to Regeneration Sugarcane Medium RSCM-25 (4.3 g/l MSsalts and vitamins supplemented with 20 g/l sucrose, 0.5 g/l casein, 0.5mg/l kinetin. pH to 5.8 and solidify with 2 g/L GELRITE®; furthersupplemented with 25 mg/l G418 sulfate after autoclaving) in low light(16 hour day length, 26° C.) to initiate regeneration. Simultaneous withinitiating regeneration, this callus tissue was evaluated by PCR forpresence of the genes on the MC. PCR confirmed that these genes wereindeed present in the majority of the events. After 2 additional weeksof culture, the callus events had started to differentiate intoplantlets (shoot material).

After an additional 4-6 weeks on regeneration, plantlets (with andwithout initial root initiation) were transferred to Rooting MediumRtSC-25 (2.15 g MS salts and vitamins supplemented with 20 g/l sucrose,0.5 g/l casein, 0.5 mg/l kinetin. pH to 5.8 and solidify with 2 g/LGELRITE®; further supplemented with 25 mg/l G418 sulfate afterautoclaving). Rooting occurred in 2 to 6 additional weeks of culturewith 16 hour day length at 26° C. in sundae cups (Solo Cup Company).Plantlets with well established root systems were transferred intopre-moistened soil-less mix (LC1, BFG Supply Company) under a humidomein 18-well flats in a growth chamber (28° C., 16 hour day length). Thedome was opened slightly 3-4 days after transplanting to slowly reducehumidity. The dome was removed completely 2 days later and the plantletswere then transferred to a greenhouse (28° C., 16 hour day length). Theplants were watered from trays beneath the pots when the soil began todry. The plants were subsequently transplanted and grown to maturity in1.6 gallon pots with Soil:Peat:Perlite (1:1:1) supplemented withOSMOCOTE® fertilizer (The Scotts Miracle-Gro Company; Marysville, Ohio;USA).

Sugarcane callus and tissues produced phenolic compounds while in tissueculture, and these phenolic compounds appeared to reduce or inhibitcallus growth and plantlet regeneration. In order to promote Sugarcaneplantlet regeneration in culture, the media described above (MSO, MS3and variants thereof and RSCM) were supplemented withpolyvinylpyrrolidone (PVP) at a concentration ranging from 1% to 3% w/vaccording to the intensity of the exudation of the phenolic compounds.The PVP acted as a sink for phenolic compounds and enhanced subsequentcallus growth and plantlet regeneration.

In order to promote the frequency and the morphogenetic competence ofregenerable Sugarcane callus, the cells were cycled from a liquidculture to a solid culture. The apical region of the Sugarcane stem(20-30 cm long) was collected, and the mature leaves were removed. Thestem was surface-sterilized by submerging the tissue in a solution of 50ml bleach in 1 liter of water for 10 minutes. The remaining outermostmature leaves were aseptically removed, and the young inner immatureleaves were sliced into sections/discs approximately 2-3 mm thick.

The resulting leaf roll discs were placed on Sugarcane MS3 Medium at 28°C. for 2 weeks in the dark. The resulting regenerable Sugarcane callus(white nodular embryogenic pieces) was then removed and placed intoliquid Sugarcane MS1 medium at 28° C. for 2 weeks on a rotating orbitalshaker (100-150 rpm) in the dark. After the two week culture in theliquid MS1 medium, the regenerable Sugarcane callus (white nodularembryogenic pieces) was removed and sub-cultured back onto Sugarcane MS3Medium (MS3) at 28° C. for 2 additional weeks in the dark. Sugarcanecallus can be sub-cultured in 2-week intervals between solid MS mediumcontaining 3 mg/l 2,4-D and liquid MS medium containing 1 mg/l 2,4-D tomaintain embryogenic callus.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference. Applicantintends that the sequence listing filed herewith forms a part of thedescription of the specification and is hereby incorporated by referencein its entirety.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A polynucleotide comprising a nucleotide sequence selected from thegroup consisting of: (a) the nucleotide sequence of SEQ ID NO: 204; and(b) a nucleotide sequence that is at least 98% identical to thenucleotide sequence of SEQ ID NO:
 204. 2. The polynucleotide of claim 1,wherein the polynucleotide comprises the nucleotide sequence of SEQ IDNO:204.
 3. A nucleic acid comprising an array comprising at least twocopies of the polynucleotide of claim
 1. 4-5. (canceled)
 6. A nucleicacid comprising an array comprising from 2 to 1000 copies of thepolynucleotide of claim
 1. 7. (canceled)
 8. The nucleic acid of claim 1,wherein the array is from 1 to 200 kb in length.
 9. (canceled)
 10. Asugarcane centromere comprising the polynucleotide of claim
 1. 11.(canceled)
 12. A sugarcane artificial chromosome comprising thepolynucleotide of claim
 1. 13. (canceled)
 14. A sugarcane artificialchromosome comprising the sugarcane centromere of claim
 10. 15. Thesugarcane artificial chromosome of claim 12, wherein the sugarcaneartificial chromosome comprises an exogenous nucleic acid. 16.(canceled)
 17. The sugarcane artificial chromosome of claim 15, whereinat least one exogenous nucleic acid is operably linked to a heterologousregulatory sequence functional in a sugarcane plant cell. 18-19.(canceled)
 20. A vector or cell comprising the polynucleotide ofclaim
 1. 21-22. (canceled)
 23. A vector or cell comprising the sugarcaneartificial chromosome of claim
 12. 24-27. (canceled)
 28. A cellcomprising the vector of claim
 20. 29-33. (canceled)
 34. A sugarcaneplant tissue or sugarcane plant comprising the plant cell of claim 28.35-36. (canceled)
 37. A sugarcane seed obtained from the sugarcane plantof claim
 28. 38. A sugarcane plant progeny comprising a sugarcaneartificial chromosome, wherein the plant progeny is the result ofbreeding a plant of claim 34 comprising the sugarcane artificialchromosome.
 39. A method of using a sugarcane plant of claim 34, whereinthe sugarcane plant comprises a sugarcane artificial chromosomecomprising an exogenous nucleic acid encoding a recombinant protein, themethod comprising growing the plant to produce the recombinant protein.40. The method of claim 39, further comprising the step of harvesting orprocessing the sugarcane plant.